CAS12A SYSTEM FOR COMBINATORIAL TRANSCRIPTIONAL REPRESSION IN EUKARYOTIC CELLS

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant Number R01 HG012227 awarded by the National Institutes of Health and. The government has certain rights in the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63/424,050 filed November 9, 2022, the filing date of United States Provisional Patent Application Serial No. 63/447,997 filed February 24, 2023, and the filing date of United States Provisional Patent Application Serial No. 63/538,188 filed September 13, 2023, the disclosure of which applications are herein incorporated by reference.

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0003] The contents of the electronic sequence listing (UCSF-697WO_Seq_List.xml; Size: 33,258 bytes; and Date of Creation: October 27, 2023) is herein incorporated by reference in its entirety

INTRODUCTION

[0004] Bacterial adaptive immune systems employ CRISPRs (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins for RNA-guided nucleic acid cleavage. The CRISPR-Cas systems thereby confer adaptive immunity in bacteria and archaea via RNA-guided nucleic acid interference. To provide anti-viral immunity, processed CRISPR array transcripts (crRNAs) assemble with Cas protein-containing surveillance complexes that recognize nucleic acids bearing sequence complementarity to the virus derived segment of the crRNAs, known as the spacer. Class 2 CRISPR-Cas are streamlined versions in which a single Cas protein bound to RNA is responsible for binding to and cleavage of a targeted sequence. The programmable nature of these minimal systems has facilitated their use as a versatile technology for genome editing. Provided herein are methods, nucleic acids, recombinant expression vectors and systems for transcriptionally silencing genomic target sites in a cell SUMMARY

[0005] The present disclosure provides methods for transcriptionally modulating genomic target sites in a cell comprising contacting a cell with: a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides, wherein the variant type

V CRISPR/Cas effector polypeptide comprises a nickase mutation and the one or more heterologous polypeptide comprise a transcriptional modulation domain; and one or more guide RNAs (gRNA), thereby transcriptionally modulating genomic target sites in the cell.

[0006] The present disclosure provides methods for for epigenetically modifying genomic target sites in a cell comprising contacting the cell with a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides, wherein the variant type

V CRISPR/Cas effector polypeptide comprises a nickase mutation and the heterologous polypeptide is an epigenetic modification domain; and one or more guide RNAs (gRNA), epigenetically modifying genomic target sites in the cell.

[0007] The present disclosure also provides nucleic acids, recombinant expression vectors, and systems comprising the first and second expression cassettes of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

[0008] FIG. 1A-FIG. IE dAsCas!2a-KRAB variants are dose-limited and hypoactive in lentivirally delivered CRISPRi activity, despite incorporating state-of-the- art optimizations. A) Schematic for assaying CRISPRi activity of Casl2a constructs using lentivirally transduced single-plex or 3-plex crRNAs targeting cell surface marker genes assayed by antibody staining and flow cytometry. B) K562 cells constitutively expressing dAsCasl2a- KRABx3 (Campa et al., 2019) were lentivirally transduced with single crRNAs targeting CD55, CD81, B2M, KIT, or a non-targeting crRNA, and assayed by flow cytometry 6 days after crRNA transduction. C) A panel of Casl2a variants harboring novel combinations of mutations are tested using crCD55-4 and crCD81-l using the fusion protein domain architecture shown. Both Casl2a fusion protein and crRNA constructs are delivered by lentiviral transduction. D908A is a mutation in the RuvC catalytic triad that renders Casl2a DNase-inactive (Yamano et al., 2016; Zetsche et al., 2015). Other mutations are described in detail in the main text. Target gene expression knockdown was quantified by flow cytometry 6 days after crRNA transduction. D) Analysis of CD81 knockdown in cells lentivirally transduced with denAsCasl2a-KRAB protein construct at MOI ~1 vs. MOI ~5. while maintaining constant crRNA MOI (<0.74) for each crRNA construct. E) Same as D, but maintaining constant denAsCasl2a-KRAB protein construct MOI at ~5, while crRNA MOI is varied as indicated. All flow cytometry data are shown in B-E were obtained 6 days after crRNA transduction. Shown are single-cell distributions of cell surface protein expression assayed by flow cytometry 6 days after crRNA transduction. Target gene expression knockdown is shown as a percentage of non-targeting control, with Y-axis tick marks displayed with log2 spacing. Median and interquartile range are shown for each distribution. Percentage of cells below the 5th percentile (dashed line) of non-targeting crRNA are shown. Where relevant, one-sided Wilcoxon rank-sum test was performed on single-cell distributions for each replicate (asterisk indicates p<0.01).

[0009] FIG. 2A-FIG. 2F multiAsCas!2a-KRAB (R1226A/E174R/S542R/K548R), an engineered variant that favors a nicked DNA intermediate, substantially improves lentivirally delivered CRISPRi activity. A) Hypothesis of R1226A’s impact on the chromatin residence time and CRISPRi activity of AsCasl2a-KRAB fusion proteins supported by prior in vitro studies, as detailed in the main text. Sizes of arrows qualitatively reflect rates of biochemical steps. B) Comparison of denCasl2a-KRAB (D908A/E174R/S542R/K548R) vs. multiAsCasl2a-KRAB (R1226A/E174R/S542R/K548R) in CRISPRi knockdown of CD81 using crCD81-l . Left panel: Holding crRNA MOI constant at ~3 while testing protein MOI ~1 vs. ~5. Right panel: Holding protein MOI constant at ~5 while testing crRNA MOI at ~3 vs. ~0.5. Asterisks indicate p <0.01 for one-sided Wilcoxon rank-sum test of single-cell distributions. One biological replicate is shown for each condition; additional replicates shown in Fig. S9. C) Comparison of CD81 knockdown by lentivirally delivered denAsCasl2a-KRAB vs. multiAsCasl2a-KRAB at protein MOI ~1 vs. ~5 across a panel of single and 3-plex crRNA constructs, while holding constant crRNA MOI for each paired fusion protein comparison for each crRNA construct. Dashed gray line indicates 5th percentile of non-targeting crRNA control. crRNA MOI indicated by color scale. Lines connect paired replicates. One-sided Wilcoxon rank-sum tests were performed on single-cell distributions for each replicate, and asterisk denotes p<0.01 for all paired replicates within each condition. Dots indicate flow cytometry measurement 10 days after crRNA transduction; triangles indicate flow cytometry measurement 16 days after crRNA transduction. D) Same as C but showing scatter plot of CD55-APC and CD81-PE antibody co-staining signals on flow cytometry performed 16 days after transduction of the indicated crRNA constructs. Quadrants drawn based on the 5th percentile of non-targeting controls and the percentage of cells in each quadrant denoted. E) K562 cells piggyBac-engineered to constitutively express denAsCasl2a-KRAB or multiAsCasl2a-KRAB were transduced with the in-dicated crRNA constructs, followed by measurement of CD151 expression by antibody staining and flow cytometry 13 days after crRNA transduction. Median CD 151 expression knockdown relative to non-targeting control is shown for each individual replicate. Dashed gray line indicates 5th percentile of non-targeting crRNA control. crRNA MOI indicated by color scale. F) Indel quantification from PCR amplicons surrounding target sites of crCD81-l and crCD55-4 in cells lentivirally transduced at protein MOI ~5 for denAsCasl2a-KRAB and multiAsCasl2a-KRAB. Cells lentivirally transduced with opAsCasl2a (DNase fully active) are shown for comparison. Percent of reads containing indels at each base position46within the amplicon is plotted, with labels indicating maximum indel frequency observed across all bases within the amplicon.

[0010] FIG. 3A-FIG. 3H multiAsCasl2a enables combinatorial transcriptional repression using up to 6-plex crRNA constructs delivered by lentiviral transduction. All protein constructs shown were delivered by piggyBac transposition into K562 cells and sorted for the same expression level of the P2A-BFP marker (except opAsCasl2a was delivered by lentiviral transduction and selected for by puromycin-resistance marker). A) Schematic for higher-order (>3-plex) crRNA expression constructs. 23nt spacers are interspersed by 19nt direct repeat variants (DeWeirdt et al., 2020) uniquely assigned to each position within the array. B) Flow cytometry analysis of CD81 expression knockdown by antibody staining 6 days after transduction of the indicated lentiviral crRNA constructs in K562 cells engineered to constitutively express the indicated panel of fusion protein constructs. Shown are averages of me-dian single-cell expression knockdown from 2-5 biological replicates for each crRNA construct, with error bars indicating SEM. One-sided Wilcoxon rank-sum test was performed for differences in single-cell expression distributions for each fusion protein against multiAsCasl2a-KRAB for each individual replicate. Asterisk indicates p < 0.01 for all replicates for a given pairwise comparison. C) Same as B, but shown for KIT expression knockdown. D) Indel quantification for the indicated fusion protein constructs using a 6-plex crRNA construct encoding crKIT-2 and crKIT-3 that target opposite strands at sites spaced 95bp apart near- the KIT TSS. Following crRNA transduction, cells were sorted on day 3 for GFP marker on the crRNA construct, and the 340bp genomic region surrounding both crRNA binding sites was PCR amplified from cell lysates harvested 15 days after crRNA transduction. The maximum percentages of reads containing indels overlapping any base position within each of the demarcated regions (region A, region B, region C) are shown. E) Comparison of the indicated fusion protein constructs in dual CD55 and CD81 CRISPRi knockdown 10 days after lentiviral transduction of a 6-plex crRNA construct by flow cytometry. Shown are log 10 fluorescence intensity for each antibody stain and the percentages of cells in each quadrant, defined by the 5th percentile of non-targeting crRNA for each fluorescence signal, are indicated. F) Summary of the same experiment in E for a larger panel of crRNA constructs, showing the percentage of cells with successful double-knockdown of CD55 and CD81 (e.g. same gating strategy as bottom left quadrant in E). G) Analogous to F, except triple knockdown of CD55, KIT, and CD81 was quantified by the percentage of cells that are below the 5th percentile along all 3 dimensions on day 33 after transduction of crRNA constructs. H) Gene expression knockdown by multiAsCasl2a-KRAB using 6-plcx, 8-plcx and 10-plcx crRNA array constructs was measured by flow cytometry 10-11 days after lentiviral transduction of crRNA constructs.

[0011 ] FIG. 4A-FIG. 4G multiAsCasl2a-KRAB enables TSS-targeting pooled CRISPRi screens, including with 6-plex crRNA arrays. A) Design of Library 1. B) Library 1: Scatter plot of cell fitness scores in K562 cells for multiAsCasl2a-KRAB vs. denCasl2a-KRAB for 3,334 single crRNA constructs with sufficient read coverage for analysis and targeting canonical TTTV PAMs within -50bp to +300bp window of 584 essential gene TSS’s. Marginal histograms show percentage of crRNA constructs with cell fitness scores exceeding the 5th percentile of negative control crRNAs. C) 2D density plots of cell fitness scores vs. predicted crRNA on-targeting efficacy score from the CRISPick algorithm, grouped by TTTV PAM vs. non-canonical PAM’s. The 5th percentiles of intergenic negative control crRNAs cell fitness scores are shown as a dashed horizontal line and the percentage of crRNAs below that threshold shown in the marginal histogram. Pearson correlation coefficient shown. D) Library 1: Moving average cell fitness score across all TTTV crRNAs at each PAM position relative to the TSS (left), shown for the 240 essential gene TSS’s for which analogous dCas9-KRAB NGG PAM tiling screen data (Nu~nez et al., 2021) is available in K562 cells (right). E) Library 1: Boxplots of average cell fitness scores of top 3 crRNAs for each essential TSS for multi AsCasl 2a- KRAB or denCas 12a- KRAB, subtracted by the average cell fitness scores from top 3 sgRNAs for the same TSS for dCas9-KRAB (Nu~nez et al., 2021). Boxplots show median, interquartile range, whiskers indicating 1.5x interquartile range, and are overlaid with individual data points. F) Design of Library 2 Sublibrary A, aimed at evaluating CRISPRi activity at each position of 6- plex crRNA arrays in K562 cells. G) Library 2 Sublibrary A: Analysis of 66,306 6-plex constructs with sufficient read coverage and encodes in the test position one of 2,971 spacers that scored as strong hits as single crRNAs in the Library 1 screen, or an intergenic negative control. Boxplots of cell fitness scores averaged from the top 3 context constructs for each given test position in the 6-plex array, grouped by negative control spacers vs. essential TSS-targeting spacers in a given test position. Percent recall is calculated as the fraction of essential TSS- targeting spacers (that were empriically active in the single crRNA Library 1 screen) recovered by the Library 2 6-plex crRNA array screen for a given test position, using the 5th percentile of negative control spacers as a threshold for calling hits. Boxplots display median, interquartile range, whiskers indicating 1.5x interquartile range, and outliers.

[0012] FIG. 5A-FIG. 5C multiAsCasl2a-KRAB CRISPRi enables enhancer perturbation and discovery. A) K562 cells constitutively expressing multiAsCasl2a-KRAB are lentivirally transduced with single crRNAs targeting the HBG TSS or its known enhancer, HS2. Shown are HBG mRNA levels measured by RT-qPCR, normalized to GAPDH levels. B) Genome browser view of the CD55 locus, including predicted enhancers using the activity-by- contact model and DNase-seq and H3K27Ac ChlP-seq tracks from ENCODE. K562 cells piggyBac-engineered to constitutively express multiAsCasl2a-KRAB was transduced with 4- plex crRNA constructs targeting regions (Rl-Rl 1) in the CD55 locus, and R12 as a negative control region devoid of enhancer features. Each unique 4-plex crRNA construct is labeled as ”a” or ”b”. For comparison, targeting the CD55 promoter using a 6-plex crRNA array (crCD55- 4 crB2M-l crKIT-2 crKIT-3 crCD81-l) is included. CD55 expression was assayed by flow cytometry between 9 and 11 days after crRNA transduction. C) Comparison of CRISPRi targeting using multiAsCasl2a-KRAB vs. opAsCasl2a using a subset of crRNA constructs form B, plus a crRNA construct targeting a coding exon of CD55 as a positive control for knockdown by DNA cutting. CD55 expression was assayed by flow cytometry 11 days after crRNA transduction.

[0013] FIG. 6A-FIG. 6D multiAsCasl2a-KRAB enables combinatorial targeting of cis-regulatory elements in pooled CRISPRi screens. A) Genome browser view of the MYC locus, including activity-by-contact model predictions, and DNase-seq and H3K27Ac ChlP-seq tracks from ENCODE. 3 of the known MYC enhancers (el, e2, e3) in the body of the noncoding RNA, PVT1, are shown. B) K562 cells piggy Bac-engineered to constitutively express the indicated panel of fusion protein constructs were transduced with one of 4 3-plex crRNA constructs targeting the MYC promoter or co-targeting the 3 enhancers using one crRNA per enhancer. Cell fitness as a proxy of MYC expression is measured as log2 fold-change in percentage of cells expressing GFP marker on the crRNA construct, relative to day 3 after crRNA transduction. E) 6,370 6-plex permutations of the 12 individual spacers from B, together with 3 intergenic negative control spacers, were designed and cloned as 6-plex crRNA arrays used in the design of Library 2 Sublibrary B. D) Library 2 Sublibrary B: Analysis of 1,446 constructs with sufficient read coverage, categorized based on whether each contains the 3 crRNAs that target the MYC promoter (which are either present or absent simultaneously in this analysis), and/or at least one crRNA that targets each of the MYC enhancers. Boxplots show cell fitness score distributions (as proxy of MYC expression) of all constructs that fall in each category. Boxplots show median, interquartile range, whiskers indicating 1.5x interquartile range, and are overlaid with individual data points each representing a 6-plex construct.

[0014] FIG. 7 Group testing framework for efficient exploration of combinatorial CRISPR perturbations.

[0015] FIG. 8 Example of flow cytometry gating strategy for CRISPRi experiments.

General gating strategy for CRISPRi experiments using flow cytometry readouts, shown for K562 cells as an example. Single live cells are gated by FSC vs. SSC, followed by gating for the fluorescent marker on the crRNA construct (typically GFP), which are subsequently analyzed for target gene expression in the respective fluorescence channels.

[0016] FIG. 9 Additional replicate for Fig. IB. See Fig. IB for details.

[0017] FIG. 10 Western blot of fusion proteins. Western blot of whole-cell lysates prepared from K562 cells piggyBac engineered to constitutively express each of the fusion proteins in the panel. anti-HA tag was used for detection of the fusion protein and anti-GAPDH for detection of GAPDH as loading control.

[0018] FIG. 11A-FIG. 11B CRISPRi activity of multiCasl2a-KRAB, denAsCasl2a- KRAB, and dAsCasl2a-KRABx3 in C42B cells. A) C4-2B cells piggy Bac-engineered to constitutively express each of the fusion protein constructs are lentivirally transduced with the indicated crRNA constructs. Cells were sorted based on P2A-BFP marker signal. Because some of these cell lines showed slightly different levels of BFP signal as a proxy of fusion protein expression, to account for fusion protein expression we performed propensity score matching to subset for populations of cells for each fusion protein construct with the same distributions in BFP signals after data acquisition for flow cytometry in CRISPRi experiments. The BFP signals before and after matching are shown as violin blots overlaid with boxplots showing median and interquartile range. B) Target gene expression are measured by cells surface antibody staining and flow cytometry 13-14 days after crRNA transduction. Single-cell distributions of expression knockdown relative to non- targeting crRNA are shown with mean and interquartile range indicated using the cells after propensity score matching for BFP levels as described in A. CRISPRi knockdown results are indistinguishable with and without propensity score matching (not shown).

[0019] FIG. 12 dAsCasl2a-KRABx3 CRISPRi by transient transfection in HEK 293T cells. HEK 293T cells were co-transfcctcd with a plasmid encoding for dAsCasl2a- KRABx3 and plasmids encoding for the indicated crRNA constructs targeting CD55. Cells were sorted 2 days after transfection for successful co-transfection based on BFP and GFP markers on the plasmids and CD55 expression was measured by antibody staining on flow cytometry 6 days after transfection. Violin plots of single-cell distributions of CD55 expression knockdown as a percentage of the median of non-targeting control are shown. Median and interquartile range are shown in the plot. The percentage of cells below the 5th percentile of the non-targeting control are also shown.

[0020] FIG. 13A-FIG. 13C Comparisons of dAsCas!2a variant fusion CRISPRi constructs using up to 3-plex crRNA constructs. A) The same fusion protein schematic as shown in Fig. 1C, labeled with construct IDs for ease of reference. B) CD55 expression knockdown measured by flow cytometry using the indicated crRNA constructs and the panel of fusion protein constructs in A. Shown are averages of the median single-cell expression knockdown relative to non-targeting crRNA for 3 biological replicates (includ-ing the replicate for crCD55-4 shown in Fig. 1C) for all comparisons, except the comparison for crCD81-l crCD151-3 crCD55-4 contains 2 replicates. For one-sided Wilcoxon rank-sum test comparing denAsCasl2a-KRAB (pCH4) to each of the other fusion constructs in the panel was performed. Asterisk indicates p<0.01 for all replicate-level comparisons for a given construct comparison. C) Analogous to B, but for CD81 knockdown. Summaries shown for 3 biological replicates (including the replicate for crCD81-l shown in Fig. 1C) for all comparisons, except the comparison for crCD81-l crCD151-3 crCD55-4 contains 2 replicates.

[0021 ] FIG. 14A-FIG. 14B Additional replicates testing effect of dose on denAsCas!2a-KRAB. A) Summary of all replicates for experiment shown in Fig. ID: shown are averages of median expression knockdown for each crRNA construct (N = 3-6 biological replicates for each crRNA construct, including the replicate shown in Fig. ID). Error bars denote SEM. One-sided Wilcoxon rank-sum test was performed on the medians of single-cell expression knockdown of each replicate and p-values indicated where relevant. B) Second biological replicate for Fig. IE; see Fig. IE for details.

[0022] FIG. 15A-FIG. 15F Testing CRISPRi activity of lentivirally delivered truncated crRNAs. In all panels, the indicated cell line, either RN2 (FIG. 15A-FIG. 15D) or B16 (FIG. 15E and FIR. 15F) was engineered for constitutive expression of the indicated fusion protein constructs by Icntiviral transduction, followed by lentiviral transduction (at MOI between 0.3-0.4) of the indicated single-plex crRNA constructs containing spacers of the indicated lengths targeting Rpa3, an essential gene. The spacers target either the gene’s coding exon, the TSS region, or the Rosa locus (negative control) as indicated in the legends. Cell fitness phenotype over time is measured in a competition assay by quantifying log2 fold change in percent of cells expressing the GFP marker on the crRNA expression constructs. Error bars indicate SEM for N = 3 biological replicates for all panels.

[0023] FIG. 16 CD81 knockdown denAsCasl2a-KRAB vs. multiAsCasl2a-KRAB at different protein and crRNA MOIs. Second biological replicate for Fig. 2B. See Fig. 2-B for further details.

[0024] FIG. 17 CD55 knockdown by denAsCasl2a-KRAB vs. multiAsCasl2a-

KRAB at different protein MOIs. Comparison of CD55 knockdown by lentivirally delivered denAsCasl2a-KRAB vs. multiAsCasl2a-KRAB at protein MOI ~1 vs. ~5 across a panel of single and 3-plex crRNA constructs, while holding constant crRNA MOI for each paired fusion protein comparison for each crRNA construct. Dashed gray line indicates 5th percentile of nontargeting crRNA control. crRNA MOI indicated by color scale. Lines connect paired replicates. One-sided Wilcoxon rank-sum tests were performed on single-cell distributions for each replicate, and asterisk denotes p<0.01 for all paired replicates within each condition. Dots indicate flow cytometry measurement 10 days after crRNA transduction; triangles indicate flow cytometry measurement 16 days after crRNA transduction.

[0025] FIG. 18A-FIG. 18C RNA-seq analysis of crRNA specificity. A) K562 cells lentivirally engineered (MOI ~5) to constitutively express multiAsCasl2a-KRAB were either transduced with the indicated crRNA’s at MOI <0.3, followed by sorting for crRNA-transduced cells based on GFP marker, or received no crRNAs. RNA was isolated from the sorted cells 32 days of culture after crRNA transduction and subjected to 3’ RNA-seq. Scatter plot of normalized mRNA expression levels for crRNA transduced (1 biological replicate each) vs. cells without crRNA (2 biological replicates), and Pearson correlation coefficient calculated for the transcriptome, excluding HBG. RT-qPCR quantifications are shown in Fig. 5. B) Volcano plots of differential expression analysis from DE-seq2, and genes that fall beyond p-value and log2FoldChange cutoffs (dashed lines) highlighted. C) Lists of differentially expressed genes (other than HBG) in the crHBG-3 and crHS2-3 transduced cells are shown. For comparison, a list of all off-target predictions generated by crisprVerse are shown for all crRNAs in the panel in A and B.

[0026] FIG. 19A-FIG. 19B CRISPRi knockdown of CD55 and B2M using up to 6- plex crRNA arrays. Same as Fig. 3B-C, shown for CD55 (FIG. 19A) and B2M (FIG. 19B) knockdown on day 6 after crRNA transduction, measured by antibody staining of those targets using flow cytometry. Shown are averages of median single-cell expression knockdown from 2- 5 biological replicates for each crRNA construct, with error bars indicating SEM.

[0027] FIG. 20A-FIG. 20B Monitoring P2A-BFP reporter as proxy of fusion protein expression level. A) K562 cells lentivirally engineered (at MOI ~1 or MOI ~5) to constitutively express the indicated fusion protein constructs were monitored for P2A-BFP expression levels by flow cytometry. B) Same as A for K562 cells piggyBac-engineered to constitutively express the indicated fusion protein constructs. opAsCasl2a does not contain BFP reporter and is shown as fluorescence negative control.

[0028] FIG. 21A-FIG. 21B Indel quantification and gene expression knockdown simulation for dual-targeting of the KIT TSS region. A) Indel quantification of PCR amplicon near the KIT TSS region in K562 cells lentivirally engineered to constitutively express opAsCasl2a 15 days after transduction of the indicated 6-plcx crRNA array (sorted for crRNA transduced cells on 2 days after transduction). Note that opAsCasl2a is encoded in a different expression backbone using a puromycin selectable marker and thus is not directly matched to other fusion constructs in Fig. 3 in transgenic expression level. B) Based on the observed indel allelic frequencies in A and Fig. 3D, we calculated the expected proportion of cells that harbor a specified number of DNA copies containing indels of any size within the PCR amplicon, assuming indels induced by crKIT-2 and crKIT-3 occur independently across DNA copies within each cell (see Methods). Based on these proportions we simulated the expected distribution of single-cell gene expression levels under the assumption that knockdown were solely due to genetically null deletions of any size abolishing KIT expression in cis (“expected null”). Expected knockdown under this genetic null assumption exceeds that observed for opAsCasl2a (fully active DNase), demonstrating the genetic null assumption is an overestimate of gene expression effects of indels in this region. To correct for this overestimate, we use the ratio of observed vs. expected null median expression knockdown by opAsCasl2a as an estimate of the hypomorphic effect of deletions in this region (“hypomorphic coefficient”). We multiply the expected null median expression knockdown for all other fusion proteins by this hypomorphic coefficient to obtain an “expected hypormorph” median expression knockdown, which we propose as our final estimate of the effects arising from indels. The observed knockdown values are the same as shown in FIG. 3D.

[0029] FIG. 22A-FIG. 22B Indel quantification and gene expression knockdown simulation for single-targeting of the KIT TSS region. Analogous to FIG. 3D and FIG. 21, but for a single-site targeting of the KIT TSS region using crKIT-2 encoded within a 4-plex crRNA array.

[0030] FIG. 23A-FIG. 23B Double and triple gene knockdown by CRISPRi using higher-order crRNA arrays. A) Single-cell view of CD81, KIT, and CD55 3-way knockdown using a 6-plex crRNA construct in K562 cells piggyBac-engineered to constitutively express each of the indicated fusion protein constructs, measured by multiplexed flow cytometry. Summary of percentage of cells with triple knockdown is shown in Fig. 3-G. B) Quantification of the fraction of cells showing double -knockdown of pairs of target genes in the experiment described in A for 4-plex and 6-plex crRNA arrays. Double knockdown is defined as the fraction of cells with expression below the 5th percentile of non-targeting crRNA for a given pair of target genes. [0031 ] FIG. 24A-FIG. 24C Summaries of Library 1 and Library 2 screens. A)

Summary of crRNA constructs in the Library 1 screen. B) Summary of crRNA constructs in the Library 2, Sublibrary A screen. C) Summary of crRNA constructs in the Library 2, Sublibrary B screen.

[0032] FIG. 25A-FIG.25B Screen replicate concordance for Library 1 and Library

2. Shown are 2D density plots of cell fitness scores for individual crRNA constructs in A) Library 1 and B) Library 2 (Sublibrary A and Sublibrary B), with the Pearson correlation coefficients calculated for all constructs in each library shown.

[0033] FIG. 26A-FIG. 26B Cell fitness score distributions of intergenic vs. nontargeting negative control crRNAs. Boxplots of cell fitness scores for A) Library 1 single- crRNA constructs, and B) Library 2 6-plex constructs, categorized by whether the construct encodes exclusively intergenic vs. non-targeting negative control crRNAs. Boxplots display median, interquartile range, whiskers indicating 1.5x interquartile range, and outliers.

[0034] FIG. 27 Cell fitness score distributions of intergenic vs. non-targeting negative control crRNAs. Boxplots of cell fitness scores for A) Library 1 single-crRNA constructs, and B) Library 2 6-plex constructs, categorized by whether the construct encodes exclusively intergenic vs. non-targeting negative control crRNAs. Boxplots display median, interquartile range, whiskers indicating 1.5x interquartile range, and outliers.

[0035] FIG. 28 Additional data demonstrating multiplexed target gene knockdown using 10-plex crRNA array. K562 cells lentivirally transduced to constitutively express multiAsCasl2a-KRAB were transduced with the indicated 10-plex TSS-targeting crRNA array, followed by flow cytometry to assay for CD55 and CD81 expression by antibody staining. Shown are single-cell distributions log 10 fluorescence signal as scatterplot.

[0036] FIG. 29A-FIG. 29B multiAsCasl2a supports more robust transcriptional repression memory engineering using CRISPRoff. A) HEK 293T cells harboring a CLTA- GFP knock-in reporter allele were lentivirally transduced with crRNAs at low MOI, sorted for crRNA-expression cells, transiently transfected with plasmids encoding the indicated fusion protein constructs, followed by sorting for transfected cells based the BFP reporter 2 days after transfection. CRISPRoff refers to the combination of Dnmt3a, Dnmt3L and KRAB as described in Nunez et al., 2021. B) Monitoring of CLTA-GFP expression over time based on experimental workflow in A for a panel of indicated Acidaminococcus or Lachnospiraceae Casl2a fusion protein constructs.

[0037] FIG. 30 Comparison multiAsCasl2a CRISPRoff gene repression activity to a panel of other fusion proteins. Same experiment as described in FIG. 29 shown for CLTA- GFP expression assayed by flow cytometry day 6 after protein construct plasmid transfection for a larger panel of fusion proteins as indicated.

DEFINITIONS

[0038] The terms “polynucleotide” and “nucleic acid,” used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, terms “polynucleotide” and “nucleic acid” encompass singlestranded DNA; double- stranded DNA; multi- stranded DNA; single- stranded RNA; doublestranded RNA; multi- stranded RNA; genomic DNA; cDNA; DNA-RNA hybrids; and a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0039] By "hybridizable" or “complementary” or “substantially complementary" it is meant that a nucleic acid (e.g. RNA, DNA) comprises a sequence of nucleotides that enables it to non-covalently bind, i.e. form Watson-Crick base pairs and/or G/U base pairs, “anneal”, or “hybridize,” to another nucleic acid in a sequence-specific, antiparallel, manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and/or in vivo conditions of temperature and solution ionic strength. Standard Watson-Crick base-pairing includes: adenine/adenosine) (A) pairing with thymidine/thymidine (T), A pairing with uracil/ uridine (U), and guanine/guanosine) (G) pairing with cytosine/cytidine (C). In addition, for hybridization between two RNA molecules (e.g., dsRNA), and for hybridization of a DNA molecule with an RNA molecule (e.g., when a DNA target nucleic acid base pairs with a guide RNA, etc.): G can also base pair with U. For example, G/U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. Thus, in the context of this disclosure, a G (e.g., of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule; of a target nucleic acid (e.g., target DNA) base pairing with a guide RNA) is considered complementary to both a U and to C. For example, when a G/U base-pair can be made at a given nucleotide position of a protein-binding segment (e.g., dsRNA duplex) of a guide RNA molecule, the position is not considered to be non-complementary, but is instead considered to be complementary.

[0040] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The greater the degree of complementarity between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. Typically, the length for a hybridizable nucleic acid is 8 nucleotides or more (e.g., 10 nucleotides or more, 12 nucleotides or more, 15 nucleotides or more, 20 nucleotides or more, 22 nucleotides or more, 25 nucleotides or more, or 30 nucleotides or more).

[0041 ] It is understood that the sequence of a polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure, a ‘bulge’, and the like). A polynucleotide can comprise 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, 99% or more, 99.5% or more, or 100% sequence complementarity to a target region within the target nucleic acid sequence to which it will hybridize. For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. The remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined using any convenient method. Example methods include BLAST programs (basic local alignment search tools) and PowerBLAST programs (Altschul et al., J. Mol. Biol., 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), e.g., using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). [0042] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein, and refer to a polymeric form of amino acids of any length, which can include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones.

[0043] "Binding" as used herein (e.g. with reference to an RNA-binding domain of a polypeptide, binding to a target nucleic acid, and the like) refers to a non-covalent interaction between macromolecules (e.g., between a protein and a nucleic acid; between a guide RNA and a target nucleic acid; and the like). While in a state of non-covalent interaction, the macromolecules are said to be “associated” or “interacting” or “binding” (e.g., when a molecule X is said to interact with a molecule Y, it is meant the molecule X binds to molecule Y in a non- covalent manner). Not all components of a binding interaction need be sequence- specific (e.g., contacts with phosphate residues in a DNA backbone), but some portions of a binding interaction may be sequence-specific. Binding interactions are generally characterized by a dissociation constant (Ka) of less than 10’ ⁶ M, less than 10’ ⁷ M, less than 10’ ⁸ M, less than 10’ ⁹ M, less than IO ¹⁰ M, less than 10 ¹¹ M, less than 10 ^-12 M, less than 10 ^-13 M, less than 10 ^-14 M, or less than 10 ¹⁵ M. "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower Kd.

[0044] By "binding domain" it is meant a protein domain that is able to bind non- covalently to another molecule. A binding domain can bind to, for example, an RNA molecule (an RNA-binding domain) and/or a protein molecule (a protein-binding domain). In the case of a protein having a protein-binding domain, it can in some cases bind to itself (to form homodimers, homotrimers, etc.) and/or it can bind to one or more regions of a different protein or proteins.

[0045] The term "conservative amino acid substitution" refers to the interchangeability in proteins of amino acid residues having similar side chains. For example, a group of amino acids having aliphatic side chains consists of glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains consists of serine and threonine; a group of amino acids having amide containing side chains consisting of asparagine and glutamine; a group of amino acids having aromatic side chains consists of phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains consists of lysine, arginine, and histidine; a group of amino acids having acidic side chains consists of glutamate and aspartate; and a group of amino acids having sulfur containing side chains consists of cysteine and methionine. Exemplary conservative amino acid substitution groups are: valine- leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine-glycine, and asparagine-glutamine. Coded amino acids (followed in parentheses by their corresponding three-letter codes and one-letter codes) include: alanine (Ala; A), arginine (Arg; R), asparagine (Asn; N), aspartic acid (Asp; D), cysteine (Cys; C), glutamic acid (Glu; E). glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F); proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), or valine (Vai; V)

[0046] A polynucleotide or polypeptide has a certain percent "sequence identity" to another polynucleotide or polypeptide, meaning that, when aligned, that percentage of bases or amino acids are the same, and in the same relative position, when comparing the two sequences. Sequence identity can be determined in a number of different ways. To determine sequence identity, sequences can be aligned using various methods and computer programs (e.g., BLAST, T-COFFEE, MUSCLE, MAFFT, Phyre2, etc.), available over the world wide web at sites including ncbi.nlm.nili.gov/BLAST, ebi.ac.uk/Tools/msa/tcoffee/, ebi.ac.uk/Tools/msa/muscle/, mafft.cbrc.jp/alignment/software/, http://www.sbg.bio.ic.ac.uk/~phyre2/. See, e.g., Altschul et al. (1990), J. Mol. Bioi. 215:403-10.

[0047] The terms "DNA regulatory sequences," "control elements," and "regulatory elements," used interchangeably herein, refer to transcriptional and translational control sequences, such as promoters, enhancers, polyadenylation signals, terminators, protein degradation signals, and the like, that provide for and/or regulate transcription of a non-coding sequence (e.g., guide RNA) or a coding sequence (e.g., protein coding) and/or regulate translation of an encoded polypeptide.

[0048] As used herein, a "promoter sequence" is a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes. Various promoters, including inducible promoters, may be used to drive the various nucleic acids (e.g., vectors) of the present disclosure. [0049] The term "naturally-occurring" or “unmodified” or “wild type” as used herein as applied to a nucleic acid, a polypeptide, a cell, or an organism, refers to a nucleic acid, polypeptide, cell, or organism that is found in nature.

[0050] "Recombinant," as used herein, means that a particular nucleic acid (DNA or RNA) is the product of various combinations of cloning, restriction, polymerase chain reaction (PCR) and/or ligation steps resulting in a construct having a structural coding or non-coding sequence distinguishable from endogenous nucleic acids found in natural systems. DNA sequences encoding polypeptides can be assembled from cDNA fragments or from a series of synthetic oligonucleotides, to provide a synthetic nucleic acid which is capable of being expressed from a recombinant transcriptional unit contained in a cell or in a cell-free transcription and translation system. Genomic DNA comprising the relevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of nontranslated DNA may be present 5' or 3' from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms (see "DNA regulatory sequences", above). Alternatively, DNA sequences encoding RNA (e.g., guide RNA) that is not translated may also be considered recombinant. Thus, e.g., the term "recombinant" nucleic acid refers to one which is not naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a codon encoding the same amino acid, a conservative amino acid, or a non-conservative amino acid. Alternatively, it is performed to join together nucleic acid segments of desired functions to generate a desired combination of functions. This artificial combination is often accomplished by either chemical synthesis means, or by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques. When a recombinant polynucleotide encodes a polypeptide, the sequence of the encoded polypeptide can be naturally occurring (“wild type”) or can be a variant (e.g., a mutant) of the naturally occurring sequence. Thus, the term "recombinant" polypeptide does not necessarily refer to a polypeptide whose sequence does not naturally occur. Instead, a “recombinant” polypeptide is encoded by a recombinant DNA sequence, but the sequence of the polypeptide can be naturally occurring (“wild type”) or non-naturally occurring (e.g., a variant, a mutant, etc.). Thus, a "recombinant" polypeptide is the result of human intervention, but may have a naturally occurring amino acid sequence.

[0051] A "vector" or “expression vector” is a replicon, such as plasmid, phage, virus, or cosmid, to which another DNA segment, i.e. an “insert”, may be attached so as to bring about the replication of the attached segment in a cell.

[0052] An “expression cassette” comprises a DNA coding sequence operably linked to a promoter. "Operably linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. [0053] The terms “recombinant expression vector,” or “DNA construct” are used interchangeably herein to refer to a DNA molecule comprising a vector and one insert. Recombinant expression vectors are usually generated for the purpose of expressing and/or propagating the inscrt(s), or for the construction of other recombinant nucleotide sequences. The insert(s) may or may not be operably linked to a promoter sequence and may or may not be operably linked to DNA regulatory sequences.

[0054] “Heterologous,” as used herein, refers to a nucleotide or polypeptide sequence that is not found in the native nucleic acid or protein, respectively. For example, relative to a variant type V CRISPR/Cas effector polypeptide of the present disclosure, a heterologous polypeptide comprises an amino acid sequence from a protein other than the variant type V CRISPR/Cas effector polypeptide. As another example, a variant type V CRISPR/Cas effector polypeptide of the present disclosure can be fused to an active domain from a non-CRISPR/Cas effector protein (e.g., a histone deacetylase), and the sequence of the active domain could be considered a heterologous polypeptide (it is heterologous to the variant type V CRISPR/Cas effector polypeptide). As another example, a guide sequence of a guide RNA that is heterologous to a protein-binding sequence of a guide RNA is a guide sequence that is not found in nature together with the protein-binding sequence.

[0055] Acidaminococcus sp.BV3L6 Casl2a as used herein refers to the following amino acid sequence:

MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPI IDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNA IHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENA LLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLI TAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISR EAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEE FKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSA LCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKE LSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDW FAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNF QMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKT SEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEIT KEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKT TSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQ IYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRM KRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARAL LPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKE HPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVA ARQAWS VVGTIKDLKQGYLS QVIHEIVDLMIHYQ A VV VLENLNFGFKS KRT GIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKM GTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYD VKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKR IVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAID TMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDAD ANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRN (SEQ ID NO: 1)

[0056] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference.

[0057] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0058] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0059] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0060] It must be noted that as used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a variant type V CRISPR/Cas effector polypeptide” includes a plurality of such CRISPR/Cas effector polypeptides and reference to “the guide nucleic acid” includes reference to one or more guide nucleic acid and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0061 ] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0062] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

[0063] The present disclosure provides methods for transcriptionally silencing genomic target sites comprising contacting a cell with a first expression cassette comprising a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptide wherein the variant type V CRISPR/Cas effector polypeptide has a R1226A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide and the heterologous polypeptide contains a transcriptional repression domain, and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis vims post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA). The present disclosure provides methods for transcriptionally activating genomic target sites. The present disclosure provides methods for epigenetically modifying genomic target sites. The present disclosure also provides nucleic acids, recombinant expression vectors, and systems comprising the first and second expression cassettes of the present disclosure.

METHODS FOR TRANSCRIPTIONALLY MODULATING, SILENCING, ACTIVATING, AND EPIGENETICALLY MODIFYING GENOMIC TARGET SITES

[0064] The present disclosure provides methods for transcriptionally silencing genomic target sites comprising contacting a cell with a first expression cassette comprising a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptide wherein the variant type V CRISPR/Cas effector polypeptide has a nickase mutation and the heterologous polypeptide contains a transcriptional repression domain, and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA).

[0065] The present disclosure provides methods for transcriptionally activating genomic target sites comprising contacting a cell with a first expression cassette comprising the variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide wherein the variant type V CRISPR/Cas effector polypeptide has a nickase mutation and the heterologous polypeptide contains a transcriptional activation domain, and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA).

[0066] The present disclosure provides methods for epigenetically modifying genomic target sites comprising contacting a cell with a first expression cassette comprising the variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide wherein the variant type V CRISPR/Cas effector polypeptide has a nickase mutation and the heterologous polypeptide contains an epigenetic modification domain, and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA).

[0067] The methods of the present disclosure employ variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide wherein the variant type V CRISPR/Cas effector polypeptide has a nickase mutation. Nickase mutations as disclosed herein refer to mutations or amino acid substitutions that result in a slowing of the catalytic efficiency of a DNase domain, such as a RuvC DNase domain, to favor a predominantly nicked DNA state. Nickase mutations are known in the art and include a substitution of the arginine (ARG;

R) at position 1226 (R1226) of the Acidaminococcus sp.BV3L6 Casl2a amino acid sequence or a corresponding position in another type V CRISPR/Cas effector polypeptide with an alanine (Ala; A; R1226A). [[INVENTORS, PLEASE PROVIDE ANY FURTHER ANIMO ACID SUBSTITUTIONS THAT YOU KNOW OF THAT WOULD PRODUCE THE DESIRED NICKASE ACTIVITY]]

[0068] The methods of the present disclosure comprise contacting a cell with a first and second expression cassette. In general, the contacting involves introducing a nucleic acid into a host cell. Methods of introducing a nucleic acid into a host cell are known in the art, and any convenient method can be used to introduce a subject nucleic acid (e.g., an expression construct/vector) into a target cell (e.g., prokaryotic cell, eukaryotic cell, plant cell, animal cell, mammalian cell, human cell, and the like). Suitable methods include, e.g., viral infection, transfection, conjugation, protoplast fusion, lipofection, electroporation, calcium phosphate precipitation, polyethyleneimine (PEI)-mediated transfection, DEAE-dextran mediated transfection, liposome-mediated transfection, particle gun technology, calcium phosphate precipitation, direct micro injection, nanoparticle-mediated nucleic acid delivery (see, e.g., Panyam et al. Adv Drug Deliv Rev. 2012 Sep 13. pii: S0169-409X(12)00283-9. doi: 10.1016/j.addr.2012.09.023 ), and the like.

[0069] In some embodiments, in place of the first expression cassette, a cell may be contacted with a nucleic acid encoding the type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide. In some embodiments, the nucleic acid is RNA. In some embodiments, in place of the first expression cassette, type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide. In some embodiments, in place of the second expression cassette, a cell may be contacted with a nucleic acid encoding the one or more gRNAs. [0070] The first expression cassette of the present disclosure may comprise any promoter deemed useful for the expression of the type V CRISPR/Cas effector polypeptide. In some cases, the promoter is a constitutively active promoter. In some cases, the promoter is a regulatable promoter. In some cases, the promoter is an inducible promoter. In some cases, the promoter is a tissue-specific promoter. In some cases, the promoter is a cell type-specific promoter.

[0071] Non-limiting examples of eukaryotic promoters (promoters functional in a eukaryotic cell) include EFla, those from cytomegalovirus (CMV) immediate early, herpes simplex virus (HSV) thymidine kinase, early and late SV40. long terminal repeats (LTRs) from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. The expression vector may also contain a ribosome binding site for translation initiation and a transcription terminator. The expression vector may also include appropriate sequences for amplifying expression. The expression vector may also include nucleotide sequences encoding protein tags (c.g., 6xHis tag, hemagglutinin tag, fluorescent protein, etc.) that can be fused to variant type V CRISPR/Cas effector polypeptide of the present disclosure, thus resulting in a fusion polypeptide.

[0072] In some cases, a nucleotide sequence encoding a variant type V CRISPR/Cas effector polypeptide of the present disclosure, or a fusion polypeptide of the present disclosure, is operably linked to an inducible promoter. In some cases, a nucleotide sequence encoding a variant type V CRISPR/Cas effector polypeptide of the present disclosure is operably linked to a constitutive promoter.

[0073] A promoter can be a constitutively active promoter (i.e., a promoter that is constitutively in an active/”ON” state), it may be an inducible promoter (i.e., a promoter whose state, active/”ON” or inactive/“OFF”, is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), it may be a spatially restricted promoter (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), and it may be a temporally restricted promoter (i.e., the promoter is in the “ON” state or “OFF” state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[0074] Suitable promoters can be derived from viruses and can therefore be referred to as viral promoters, or they can be derived from any organism, including prokaryotic or eukaryotic organisms. Suitable promoters can be used to drive expression by any RNA polymerase (e.g., pol I, pol II, pol III). Exemplary promoters include, but are not limited to the SV40 early promoter, mouse mammary tumor virus long terminal repeat (LTR) promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, a cytomegalovirus (CMV) promoter such as the CMV immediate early promoter region (CMVIE), a rous sarcoma virus (RSV) promoter, a human U6 small nuclear promoter (U6) (Miyagishi et al., Nature Biotechnology 20, 497 - 500 (2002)), an enhanced U6 promoter (e.g., Xia et al., Nucleic Acids Res. 2003 Sep 1 ;31(17)), a human Hl promoter (Hl), and the like. [0075] The first expression cassette of the present disclosure comprises a type V

CRISPR/Cas effector polypeptide fused to a heterologous polypeptide. A wild-type type V CRISPR/Cas protein, e.g., Casl2 proteins such as Cpfl (Casl2a) and C2cl (Casl2b), can promiscuously cleave non-targeted single- stranded DNA (ssDNA) once activated by binding of a target DNA (double or single stranded). For example, when a wild-type type V CRISPR/Cas effector protein (e.g., a Casl2 protein such as Casl2a, Casl2b, Casl2c, Casl2d, Casl2c, Casl2f, Casl2g, Casl2h, or Casl2i) is activated by a guide RNA, which occurs when the guide RNA hybridizes to (binds to) a target sequence of a target DNA, the protein becomes a nuclease that promiscuously cleaves ssDNAs (i.e., the nuclease cleaves non-target ssDNAs, i.e., ssDNAs to which the guide sequence of the guide RNA does not hybridize). When a type V CRISPR/Cas effector protein is activated by a guide RNA and exhibits on-target cleavage of the target ssDNA, such on-target cleavage is referred to as “cz's” cleavage (Li et al (2018) Cell Research 28:491-493). When a type V CRISPR/Cas effector protein is activated by a guide RNA and exhibits cleavage of non-target ssDNAs (ssDNAs to which the guide sequence of the guide RNA does not hybridize) such non-target cleavage is referred to as “trans” cleavage.

[0076] In some cases, a variant type V CRISPR/Cas effector polypeptide of the present disclosure comprises a substitution of the arginine (ARG; R) at position 1226 (R1226) of the Acidaminococcus sp.BV3L6 Casl2a amino acid sequence depicted in SEQ ID NO: 1, or a corresponding position in another type V CRISPR/Cas effector polypeptide with an alanine (Ala; A; R1226A). A “corresponding position” in another type V CRISPR/Cas effector polypeptide is readily determined by aligning the amino acid sequence of a type V CRISPR/Cas effector polypeptide with the Acidaminococcus sp.BV3L6 Casl2a amino acid sequence depicted in SEQ ID NO: 1. The variant type V CRISPR/Cas effector polypeptide of the present disclosure can be from any organism deemed useful. In some embodiments, the variant type V CRISPR/Cas effector polypeptide is from an organism selected from the group consisting of Lachnospiraceae bacterium, Francisella novicida, Porphyromonas macacae, Moraxella bovoculi, Thiomicrospira sp., Butyrivibrio sp., Brumimicrobium aurantiacum, Porphyromonas crevioricanis, Francisella tularensis, Eubacterium ventriosum, etc. In a preferred embodiment, the variant type V CRISPR/Cas effector polypeptide is from Acidaminococcus sp.

[0077] The variant type V CRISPR/Cas effector polypeptide may have further comprise amino acid substitutions that enhance the activity or function of the variant type V CRISPR/Cas effector polypeptide. Amino acid substitutions that enhance the activity or function of the variant type V CRISPR/Cas effector polypeptide are known in the art and have been described in, for example, Kleinstiver et al. (Nat Biotechnol. 2019 Mar;37(3):276-2820) which is specifically incorporated by reference herein. In some embodiments, the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

[0078] The variant type V CRISPR/Cas effector polypeptide of the present disclosure is fused to a heterologous polypeptide. A heterologous polypeptide to which a variant type V CRISPR-Cas effector polypeptide of the present disclosure can be fused is referred to herein as a “fusion partner.” In some cases, a variant type V CRISPR-Cas effector polypeptide of the present disclosure is fused to one or more heterologous polypeptides that has/have an activity of interest (e.g., a catalytic activity of interest, subcellular localization activity, etc.) to form a fusion protein. In some instances, the variant type V CRISPR-Cas effector polypeptide of the present disclosure is fused to two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more heterologous polypeptides.

[0079] In some cases, the fusion partner can modulate transcription (e.g., inhibit transcription, increase transcription) of a target DNA. For example, in some cases the fusion partner is a protein (or a domain from a protein) that inhibits transcription (e.g., a transcriptional repressor, a protein that functions via recruitment of transcription inhibitor proteins, modification of target DNA such as methylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). In some cases, the fusion partner is a protein (or a domain from a protein) that increases transcription (e.g., a transcription activator, a protein that acts via recruitment of transcription activator proteins, modification of target DNA such as demethylation, recruitment of a DNA modifier, modulation of histones associated with target DNA, recruitment of a histone modifier such as those that modify acetylation and/or methylation of histones, and the like). In some cases, the fusion partner is a reverse transcriptase. In some cases, the fusion partner is a base editor. In some cases, the fusion partner is a deaminase. When the fusion partner inhibits transcription, a range of different transcriptional repression domains may be used. Non-limiting examples of transcriptional repression domains include DMNT1, SET1, HDAC11, DMNT3A, SETD8, EZH2, SUV39H1, PHF19, SALI, NUE, SUVR4, KYP, DIM5, HDAC8, SIRT3, SIRT6, MESOLO4, SET8, HST2, COBB, SET-TAF1B, NCOR, MeCP2 SIN3A, HDT1, MBD2B, NIPP1, HP1A, KRAB, or any combination thereof. Transcriptional repression domains are known in the art and have been described by, for example, Yeo et al. (Nat Methods. 2018 Aug; 15(8): 611-616) which is specifically incorporated by reference herein [[INVENTORS, PLEASE INCLUDE ANY ADDITIONAL TRANSCRIPTIONAL REPRESSION DOMAINS THAT YOU WOULD LIKE LISTED HERE]]

[0080] When the fusion partner activates transcription, a range of different transcriptional activation domains may be used. Non-limiting examples of transcriptional activation domains include a VP64 domain, a p65 domain, a Rta domain, an AD2 domain, a CR3 domain, an EKLF1 domain, a GATA4 domain, a PR VIE domain, a p53 domain, a SP1, a MYOD, MEF2C, a TAX domain, a PPARy domain, a MED1 domain, a MED7 domain, a MED26 domain, a MED29 domain, a TBP domain, a GTF2H-sD domain a GTF2B domain, or any combination thereof. Transcriptional activation domains are known in the art and have been described by, for example, Chavez et al. (Nat Methods. 2015 Apr; 12(4): 326-328) which is specifically incorporated by reference herein[[INVENTORS, PLEASE INCLUDE ANY ADDITIONAL TRANSCRIPTIONAL ACTIVATION DOMAINS THAT YOU WOULD LIKE LISTED HERE]]

[0081 ] In some cases, a fusion polypeptide of the present disclosure includes a heterologous polypeptide that has enzymatic activity that modifies a target nucleic acid (e.g., nuclease activity, methyltransferase activity, demethylase activity, DNA repair activity, DNA damage activity, deamination activity, dismutase activity, alkylation activity, depurination activity, oxidation activity, pyrimidine dimer forming activity, integrase activity, transposase activity, recombinase activity, polymerase activity, ligase activity, helicase activity, photolyase activity, or glycosylase activity).

[0082] In some cases, a fusion polypeptide of the present disclosure includes a heterologous polypeptide that has enzymatic activity that modifies a polypeptide (e.g., a histone) associated with a target nucleic acid (e.g., methyltransferase activity, demethylase activity, acetyltransferase activity, deacetylase activity, kinase activity, phosphatase activity, ubiquitin ligase activity, deubiquitinating activity, adenylation activity, deadenylation activity, SUMOylating activity, deSUMOylating activity, ribosylation activity, deribo sylation activity, myristoylation activity or demyristoylation activity).

[0083] In some cases, a fusion polypeptide of the present disclosure includes a heterologous polypeptide that has epigenetic modification activity. The epigenetic modification may include heterologous polypeptides comprising a DNA methyltransferase domaain, a DNA demethylase domain, a histone mcthyltransfcrasc domain, a histone demethylase domain, and any combination thereof. Additionally, the epigenetic modification domains may be combined with any other domains disclosed herein. Non-limiting examples of epigenetic modification domains include [[INVENTORS, PLEASE INCLUDE EPIGENETIC MODIFCATION DOMAINS THAT YOU WOULD LIKE LISTED HERE]]

[0084] The second expression cassette comprises a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or guide RNAs (gRNA). The promoter of the second expression cassette may be any of the promoters described above. In some embodiments, the promotor is an EF-la promoter. The antibiotic resistance gene of the second expression cassette may be any antibiotic resistance gene that is deemed useful. Antibiotic resistance genes that find use in the present disclosure includes, without limitation, puromycin, gentamicin, rifampicin, kanamycin, spectinomycin, ampicillin, carbenicillin, bleomycin, erythromycin, tetracycline, chloramphenicol, etc. In some embodiments, the antibiotic resistance gene is resistant to puromycin.

[0085] The second expression cassette comprises a 3’ LTR. The 3’ LTR further comprises a U6 promoter and one or more guide RNAs (gRNA). The 3’LTR may comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more 10 gRNAs. The length of the U6 promoter and the one or more gRNAs in the 3’ LTR of the second expression cassette may be any length capable of being inserted into the 3’ LTR and still express the gRNAs. For instance, the length may be 400bp or more, 500bp or more, 600bp or more, 700bp or more, or 800bp or more.

[0086] The first and/or second expression cassette of the present disclosure may be contained in an expression vector. Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:77007704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; lomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al.. Virol. (1988) 166: 154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94: 1031923, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In some cases, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector.

[0087] In some embodiments, the first expression cassette is contained in a lentivirus expression vector. In some embodiments, the second expression cassette is contained in a lentivirus expression vector. In some embodiments, the first and the second expression cassettes are contained in the same lentivirus expression vector.

[0088] The methods of the present disclosure transcriptionally silence genomic target sites in a cell by contacting the cell with the first and the second expression cassette. The number of genomic target sites silenced in a cell may be equal to the number of gRNAs present in the second expression cassette. For instance, the number of genomic target sites silenced in a cell may be comprise one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more genomic target sites. In some embodiments, the results of the contacting the cell transcriptionally silence genomic target sites in a cell when the first and the second expression cassette are expressed at low levels relative to overexpression systems.

NUCLEIC ACIDS AND RECOMBINANT EXPRESSION VECTORS

[0089] In addition to the methods disclosed herein, the present disclosure also provides nucleic acids and recombinant expression vectors comprising the first and second expression cassettes discussed above.

[0090] A nucleic acid of the present disclosure is generally DNA but may also be RNA. The nucleic acids of the present disclosure comprise the first and/or the second expression cassettes as described above. In some embodiments, the nucleic acids comprises the second expression cassette comprising a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory clement (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and at least 5 guide RNAs (gRNA). In some embodiments, the nucleic acid comprises the first expression cassette comprising a promoter and a variant type V CRISPR/Cas effector polypeptide as described above. In some embodiments, the nucleic acid comprises both the first and the second expression cassettes as described above.

[0091 ] The present disclosure also provides recombinant expression vectors. The recombinant vector includes, without limitation, a plasmid, a viral vector, a cosmid an artificial chromosome, etc. In a preferred embodiment the recombinant vector is a viral vector. Suitable expression vectors include viral expression vectors (e.g. viral vectors based on vaccinia virus; poliovirus; adenovirus (see, e.g., Li et al., Invest Opthalmol Vis Sci 35:2543 2549, 1994; Borras et al., Gene Ther 6:515 524, 1999; Li and Davidson, PNAS 92:7700 7704, 1995; Sakamoto et al., H Gene Ther 5: 1088 1097, 1999; WO 94/12649, WO 93/03769; WO 93/19191; WO 94/28938; WO 95/11984 and WO 95/00655); adeno-associated virus (AAV) (see, e.g., Ali et al., Hum Gene Ther 9:81 86, 1998, Flannery et al., PNAS 94:6916 6921, 1997; Bennett et al., Invest Opthalmol Vis Sci 38:2857 2863, 1997; Jomary et al., Gene Ther 4:683 690, 1997, Rolling et al., Hum Gene Ther 10:641 648, 1999; Ali et al., Hum Mol Genet 5:591 594, 1996; Srivastava in WO 93/09239, Samulski et al., J. Vir. (1989) 63:3822-3828; Mendelson et al., Virol. (1988) 166: 154-165; and Flotte et al., PNAS (1993) 90: 10613-10617); SV40; herpes simplex virus; human immunodeficiency virus (see, e.g., Miyoshi et al., PNAS 94: 10319 23, 1997; Takahashi et al., J Virol 73:78127816, 1999); a retroviral vector (e.g., Murine Leukemia Virus, spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus); and the like. In a preferred embodiment, the recombinant expression vector is a lentivirus expression vector.

SYSTEMS

[0092] The present disclosure provides a system comprising a variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide and a recombinant expression vector comprising the second expression cassette as described above. The systems of the present disclosure are designed to transcriptionally silence genomic target sites in a cell. The variant type V CRISPR/Cas effector polypeptide comprises a R1226A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide. The variant type V CRISPR/Cas effector polypeptide may be any of the variant type V CRISPR/Cas effector polypeptides described above. The system also comprises a recombinant expression vector comprising the second expression cassette. The recombinant expression vector may be any of the recombinant expression vectors described above. In a preferred embodiment, the recombinant expression vector is a lentiviral expression vector.

Example 1

[0093] A Casl2a variant fusion protein and gRNA expression vector combination that can be used to transcriptionally silence up to 10 targeted genomic sites per cell, including when delivered at low concentrations of ribonucleoprotein expression in mammalian cells delivered by lentiviral constructs was developed. In contrast, prior work on using Casl2a for transcriptional silencing relied exclusively on transient transfection experiments where protein and gRNA components are highly overexpressed. The ability to function in the setting of low ribonucleoprotein expression enables combinatorial targeting of multiple genomic sites per cell in high-throughput sequencing based pooled screens and therapeutic delivery in vivo. The Casl2a variant fusion protein harbors a mutation (R1226A) that allows for combinatorial transcriptional silencing. Other Casl2a variants have been tested that showed no activity for transcriptional silencing in the context of low ribonucleoprotein expression levels delivered by lentivirus. In addition, a novel U6 promoter driven gRNA expression vector that can 10 gRNA spacers on a single pre-crRNA transcript was designed. This gRNA expression vector uses a CROP-seq vector design, which enables single-cell RNA-seq readout in conjunction with reading out the gRNA identities in the same single cells. This is the first demonstration that a CROP-seq vector can accommodate an insert (U6 promoter + 10-plex pre-crRNA) in the LTR as large as 773bp. Prior knowledge of lentiviral vector biology was that a CROP-seq vector could only accommodate less than a 400bp insertion in the LTR.

[0094] While the present invention has been described with reference to the specific embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims appended hereto.

Example 2

[0095] Functional interactions among combinations of genetic elements underlie many natural and engineered phenotypes (Costanzo et al., 2019; Domingo et al., 2019; Wong et al., 2016). Such interactions can often involve higher-order (3 or more) combinations of genetic elements. A notable example includes the discovery of 4 factors as the successful gain-of- function combination that can achieve reprogramming to pluripotency (Takahashi & Yamanaka, 2006). Similarly for loss-of-function experiments, combined perturbations are often necessary to discover the functions of paralogous genes that often exhibit functional redundancy (Dede et al., 2020; Ewen-Campen et al., 2017; Gonatopoulos-Poumatzis et al., 2020; Parrish et al., 2021). In the non-coding space, more than 3 cis-regulatory elements often co-regulate the transcriptional state of a given genomic locus with varying degrees of functional overlap, often requiring combinatorial perturbations to test their functional logic (Blayney et al., 2022; Blobel et al., 2021; Kvon et al., 2021; Osterwalder et al., 2018; Xie et al., 2017). In ex vivo engineering of cell therapeutics, simultaneous genomic editing of 4 loci is moving toward clinical testing (Beam Therapeutics, 2020, 2022). Thus, combined perturbation of >3 coding or non-coding genetic elements can be critical for discovering and engineering biological properties, otherwise unattainable by lower-order perturbations.

[0096] Despite the value of higher-order genetic perturbations across biological contexts, such perturbations have been generally difficult to achieve in a scalable manner, with prior systematic analyses primarily achieved in yeast (Celaj et al., 2020; Domingo et al., 2018; Kuzmin et al., 2018; Taylor & Ehrenreich, 2014, 2015). In mammalian functional genomics, pooled CRISPR screens are currently the most widely adopted and scalable approaches (Doench, 2018; Przybyla & Gilbert, 2021). Such screens use pooled oligo synthesis for one -pot cloning of a complex guide RNA library, which are delivered to a population of cells such that each cell generally receives a unique guide RNA construct. The phenotypes induced by each guide RNA construct are measured in a massively parallel fashion across the cell population by deep sequencing in a single biological sample (Doench, 2018; Przybyla & Gilbert, 2021). However, CRISPR/Cas9-bascd pooled screens using sequencing readouts have thus far been limited in multiplexing capability, with only a few studies targeting 3 genomic sites per cell (Adamson et al., 2016; Wong et al., 2015; Zhou et al., 2020). Further multiplexing using Cas9- based pooled screening is challenging due to 1) increasingly complex iterative cloning schemes for larger constructs encoding for multiple sgRNAs each expressed from a separate promoter ((Adamson et al., 2016; Wong et al., 2015; Zhou et al., 2020)), and 2) length-dependent high frequencies of recombination in sgRNA libraries that are typically delivered as lentiviral constructs (Adamson et al., 2018; Basu et al., 2008; Sack et al., 2016).

[0097] Casl2a, a member of the type V CRISPR/Cas family, has been proposed as an alternative to (d)Cas9 for genetic perturbations for its biochemical properties compatible with higher-order multiplexing. Cas 12a harbors RNase activity, separable from its DNase activity, that can process a compact primary transcript expressed from a single promoter into multiple CRISPR RNAs (crRNA), without the need for tracrRNA (Fonfara et al., 2016; Zetsche et al., 2015). The compactness of the Casl2a crRNA, individually consisting of a 19nt direct repeat and a 19-23nt spacer, enables deterministic encoding of multiple crRNAs on a given chemically synthesized oligo for single-step cloning into the plasmid vector, expressed from a single promoter (Breinig et al., 2019; Campa et al., 2019; DeWeirdt et al., 2020; Gier et al., 2020; Zetsche et al., 2017). Cas 12a has been engineered for mammalian cell applications using its DNase activity to disrupt coding gene function targeting targeting 2 or more sites in individual well-based assays (Breinig et al., 2019; Campa et al., 2019; DeWeirdt et al., 2022;

Gonatopoulos-Poumatzis et al., 2020; Kleinstiver et al., 2019; Zetsche et al., 2017) and in pooled sequencing screens (Chow et al., 2019; Dede et al., 2020; DeWeirdt et al., 2020; Gier et al., 2020; Gonatopoulos-Poumatzis et al., 2020; J. Liu et al., 2019). However, the extent of multiplexing with DNase-competent Casl2a is expected to be limited by increasing genotoxicity in many biological contexts. Even cancer cell lines can show cumulative genotoxicity with multi-site double- stranded DNA breaks (DeWeirdt et al., 2020; Meyers et al., 2017), and nontransformed cell types can be sensitive to genotoxicity from nuclease targeting of as low as one or two sites per cell (Bowden et al., 2020; Chen et al., 2021; Haapaniemi et al., 2018; Ihry et al., 2018). In principle, perturbations of genetic elements can avoid genotoxicity by using DNase- dead Cas enzymes fused to effector domains that alter the chromatin state of the targeted site, as has been successfully achieved for dCas9 CRISPRi and CRISPRa fusion proteins in pooled sequencing screens (Gilbert et al., 2013, 2014; Koncrmann et al., 2015). Moreover, compared to Cas9 nuclease screens, dCas9 CRISPRi has been shown to be more efficient at perturbing enhancers in pooled screens (Ren et al., 2021; Tycko et al., 2019), likely due to its larger genomic window of activity via the formation of repressive chromatin. Thus, a DNase-dead Cas 12a (dCasl2a) functional genomics platform capable of simultaneous chromatin perturbations at 3 or more sites per cell is highly desirable.

[0098] Despite the success of Cas 12a as a tool for DNA cleavage, no dCasl2a-based pooled screens using either transcriptional repression or activation have been reported thus far. Several studies have used dCasl2a for transcriptional repression in human cells in individual well-based assays, reporting either successful (Campa et al., 2019; Guo et al., 2022; Y. Liu et al., 2017; Nunez et al., 2021) or unsuccessful (O’Geen et al., 2017) repression of target genes. These dCasl2a transcriptional repression studies delivered crRNA plasmids exclusively by transient transfection, which introduces high copy number and expression of synthetic components, but is limited in assay throughput and cell type compatibility. In contrast, pooled sequencing screens require single-copy integration of crRNA constructs, typically achieved by lentiviral transduction at low multiplicity of infection (MOI), to ensure that cellular phenotypes can be attributed to unique crRNA constructs (Doench, 2018; Przybyla & Gilbert, 2021). Compared to transient plasmid transfections at high copy number, lentiviral transduction of crRNA constructs — especially at single-copy integration — is expected to result in much lower concentrations of functional Casl2a ribonucleoprotein in the cell, which imposes more stringent requirements on the functional potency per molecule. We noted that none of the published studies on dCasl2a-based transcriptional repression or (Campa et al., 2019; Guo et al., 2022; Y. Liu et al., 2017; Nunez et al., 2021) or activation (Campa et al., 2019; Guo et al., 2022; Kleinstiver et al., 2019; Tak et al., 2017) use lentivirally delivered crRNAs, raising the question of whether published dCasl2a constructs used for transcriptional control are sufficiently potent for high-throughput pooled sequencing screens. Engineering transcriptional repression by CRISPR-Cas is expected to require more stable chromatin occupancy than for DNA cutting applications, as the disruption of functional alleles by DNA cutting are cumulative, whereas chromatin binding events leading to transcriptional repression may not be functionally cumulative and may not occur simultaneously in the same cell at all copies of DNA, which can transcribe independently.

[0099] In this study, we show that dCasl2a fusion constructs for synthetic transcriptional repression are weak or inactive in the setting of lentivirally delivered crRNAs. Incorporating new combinations of published modifications that were shown by others to improve Casl2a DNase activity results in some improvements in dCasl2a CRISPRi activity, but is still overall deficient. Given the stark discrepancy in activity between DNase and DNase-dead applications of Casl2a, we reasoned that DNase-inactivation in dCasl2a likely destabilizes its chromatin occupancy, rendering fusion proteins ineffective for transcriptional repression. Guided by prior structural and biophysical studies of Casl2a in vitro, we hypothesized that modifying Casl2a to allow for DNA nicking while strongly disfavoring, rather than entirely eliminating, double-stranded cleavage would stabilize its chromatin occupancy and thus improve transcriptional repression. To achieve this, we engineered a new Casl2a variant that incorporates a key mutation, R1226A. This mutation was previously shown in vitro to slow the catalytic efficiency of the RuvC DNase domain to favor a predominantly nicked DNA state associated with a more stable ribonucleoproteimDNA complex (Cofsky et al., 2020), but has not been previously tested in the context of engineering transcriptional control. We show that in human cells, multiAsCasl2a fusion proteins significantly outperform existing Casl2a fusion constructs in transcriptional repression and epigenetic memory in the setting of lentivirally delivered crRNA constructs. We develop a new pooled functional genomics screening platform using multiAsCasl2a-KRAB, which increases signal-to-background ratio to critically enable high-throughput targeting and transcriptional repression of up to 6 genomic sites per cell using lentivirally delivered crRNA arrays. We demonstrate that multiAsCasl2a can be applied in higher-order combinatorial targeting of cis-regulatory elements in pooled screens, and propose a group testing framework for efficiently exploring potentially large combinatorial spaces of loss- of-function chromatin perturbations.

Results

[00100] CRISPRi using state-of-the-art dAsCas!2a fusion proteins is dose-limited and hypoactive in the setting of lentivirally delivered components. The two orthologs of Casl2a that have been most successfully used in mammalian cell applications are Acidaminococcus (As.) and Lachnospiraceae (Lb.) Casl2a. Here we focus on building a CRISPRi functional genomics platform using AsCasl2a as it is the only ortholog with demonstrated success in the published literature in DNA cutting-based pooled sequencing screens in mammalian cells (Dede et al. 2020; DeWeirdt et al. 2021; Gier et al. 2020). A previous study reported using dAsCasl2a for CRISPRi by plasmid transient transfection delivery of dAsCasl2a-KRAB 3 protein (harboring the E993A DNase-dead mutation) and crRNA in HEK 293T cells (Campa et al. 2019). To test this construct in the setting of lentivirally delivered crRNA, we introduced dAsCasl2a-KRABx3 by piggyBac transposition in K562 cells and sorted for a pool of cells stably expressing the construct, as monitored by a P2A- BFP marker. We designed single crRNA constructs targeting TTTV protospacer adjacent motifs (PAM) proximal to transcription start sites (TSS) of endogenous genes encoding cell surface proteins, whose knockdown by dCas9-KRAB has been previously successful and is fitnessneutral (Replogle et al. 2022a). Throughout this study we encoded crRNAs in a previously optimized CROP-seq (Datlinger et al. 2017) style lentiviral vector containing a U6 promoter that transcribes a pre-crRNA with a 3' direct repeat that enhances crRNA activity (Gier et al. 2020). After lentiviral transduction of single -plex crRNA constructs into K562 cells stably expressing dAsCasl2a-KRABx3, we measured target gene knockdown by flow cytometry using cell surface antibody staining (FIG. 1A) in cells gated for successful crRNA transduction (FIG. SI). We observed no expression change in any of the individually targeted genes (CD55, CD81, B2M and KIT in FIG. IB and FIG. 9). We confirmed expression of dAsCas!2a-KRABx3 by western blot (FIG. 12) and by routinely monitoring expression of the in-frame P2A-BFP transgene marker by flow cytometry. This lack of CRISPRi activity when using lentivirally transduced crRNAs is not limited to K562 cells, as the absence was similarly observed in C4-2B cells (prostate cancer cell line) stably engineered with dAsCasl2a-KRABx3 by piggyBac transposition (FIG. 13). In contrast, transient co-transfection of dAsCasl2a-KRABx3 and CD55-targeting crRNA plasmids shows modest CRISPRi knockdown in HEK 293T cells (FIG. 14), consistent with prior work (Campa et al. 2019). These findings indicate that the requirements for successful transcriptional repression using dAsCasl2a-KRABx3 in the setting of lentivirally delivered crRNA constructs are distinct from those of plasmid transient transfection delivery in HEK 293T cells used in prior studies reporting successful Casl2a CRISPRi at a few loci (Campa et al. 2019).

[00101 ] In an attempt to overcome this lack of CRISPRi activity, we tested combinations of several Casl2a mutations representing state-of-the-art optimizations of Casl2a from the literature. These include: 1) E174R/S542R/K548R (enhanced AsCasl2a, cnAsCas!2a), which are expected to contact PAM proximal DNA and have been shown to improve DNA cutting in human cells (Kleinstiver et al. 2019); 2) M537R/F870L (AsCasl2a ultra), which interact with the PAM (M537R) and the crRNA stem loop (F870L), and improve DNA cutting in human cells (Liyang Zhang et al. 2021); 3) W382A, a mutation that reduces R-loop dissociation in vitro for an orthologous enzyme (LbCasl2a W355A), but has not yet been tested in cells (Naqvi et al. 2022).

[00102] We generated six dAsCas 12a variants that each harbor the DNase-inactivating

D908A mutation, plus a select combination of the mutations described above. We cloned these variants into the same fusion protein architecture (FIG. 1C) consisting of an N-terminal 6x Myc-NLS (Gier et al. 2020) and C-terminal XTEN80-KRAB-P2A-BFP (Replogle et al. 2022) in a lentiviral expression vector. This allows us to monitor fluorescence from the P2A-BFP reporter by flow cytometry as a quantitative proxy of MOI and transgenic expression. We tested these constructs for CRISPRi activity by stable lentiviral expression of each dCasl2a variant fusion construct and a crRNA construct targeting the TSS of either CD55 and CD81 (FIG. 1C and FIG. 10) in K562 cells. Among this panel, denAsCasl2a-KRAB (E174R/S542R/K548R, plus D908A DNase-dead mutation) performed the best and demonstrated strong repression of CD55. However, even for this best construct we observed weak repression of CD81, indicating inconsistent performance across crRNAs (FIG. 1C and FIG. 10).

[00103] Dose-response and construct potency are key considerations for multiplexed applications, as increased multiplexing effectively reduces the concentration of Cas protein available to bind each individual crRNA. Focusing on denAsCasl2a-KRAB as the top variant, we tested the effect of separately altering the dosage of Cas 12a protein and crRNAs delivered. We found that increasing the MOI of the denAsCasl2a-KRAB construct from -1 to -5 can improve CRISPRi knockdown of CD81 for a single crRNA targeting CD81 (crCD81-l) and when encoded in the context of a 3-plex crRNA array in the 3' position (crCD55-4_crCD151- 3_crCD81-l), but still at a suboptimal level (-60% median expression knockdown relative to non-targeting control, FIG. ID and FIG. 11). Even at high protein construct MOI of -5, we found that CRISPRi activity of denAsCasl2a-KRAB is significantly lost when the crRNA MOI is reduced to <1 to mimic that required to ensure single-copy integrations in pooled screens using sequencing readouts (FIG. IE and FIG. 11). Even more problematically, across all protein (FIG. ID and FIG. 11) and crRNA (FIG. IE and FIG. 11) doses tested, a 3-plex crRNA in the reverse orientation (CD81-l_CD151-3_CD55-4) shows extremely weak CD81 knockdown (~0%-25% median expression knockdown relative to non-targeting control), indicating that denAsCasl2a-KRAB CRISPRi activity can be sensitive to the specific arrangement of crRNAs encoded in a multiplexed array.

[00104] Given the inconsistent and deficient performance of denAsCasl2a-KRAB, we tested an alternative CRISPRi approach without mutating the RuvC DNase domain. In the setting of transient plasmid transfection delivery in HEK 293T cells, wild-type AsCasl2a protein without any engineered RuvC DNase domain mutations has been used for transcriptional control with truncated (15nt) crRNA spacers, which enable DNA binding but not cleavage (Campa et al. 2019; Breinig et al. 2019). We tested this approach by fusing KRAB or KRABx3 in different N- and C-terminal arrangements to opAsCasl2a (containing 6x MycNLS and DNA affinity-enhancing mutations E174R/S542R), a DNase-active Cas 12a optimized for pooled screens (Gier et al. 2020). Confirming previous findings, we showed that the 15nt spacers did not support DNA cleavage, while the 23nt spacers did (FIG. 15). However, using 15nt spacers, we observed weak or no cell fitness phenotype as a proxy of CRISPRi activity when targeting the transcriptional start site of a common essential gene, Rpa3, in two cell lines engineered with a panel of opAsCasl2a fused to KRAB or 3xKRAB (FIG. 15). In total, we have tested 3 separate approaches that abolish the DNase activity of AsCasl2a: 1) E993A in dAsCasl2a-KRABx3, 2) D908A in denAsCas 12a- KRAB, and 3) use of truncated spacers with opAsCasl2a fused to KRAB or 3xKRAB. All of these CRISPRi approaches, despite incorporating state-of-the-art optimizations, perform poorly when used with lentivirally transduced crRNA constructs. These results collectively suggest additional optimization of construct potency is required for the goal of developing a robust and predictable Casl2 based CRISPRi functional genomics platform.

[00105] multiAsCas!2a-KRAB (R1226A/E174R/S542R/K548R), a variant with low DNA cleavage efficiency, substantially improves lentivirally delivered CRISPRi. The mediocre performance of dCasl2a for CRISPRi surprised us given the successful application of Casl2a in DNA-cutting pooled screens. We wondered whether full inactivation of DNA cutting in dCasl2a may render transcriptional repression ineffective by adversely impacting other aspects of protein function important for CRISPRi activity, specifically chromatin occupancy. Previous studies indicate that the interaction between Casl2a and a DNA target can be strengthened by DNA cleavage (Singh et al. 2018; Knott et al. 2019; Cofsky et al. 2020). In the Casl2a DNA cleavage pathway, a single DNase active site first cuts the non-target strand, followed by cleavage of the target strand (Swarts and Jinek 2019). While double-strand breaks are undesired for CRISPRi applications, we wondered whether favoring the intermediate nicked DNA state might reduce the R-loop dissociation rate (FIG. 2A, see Discussion). In support of this possibility, in vitro binding assays showed that dCasl2a:DNA complexes are 20-fold more stable when the non-target strand was pre-nicked (Cofsky et al. 2020), and single-molecule FRET studies suggested that non-target strand nicking biases Casl2a:DNA complexes away from dissociation-prone conformations (Zhang et al. 2019; leon et al. 2018).

[00106] To achieve nicking-induced stabilization for CRISPRi applications in mammalian cells, we incorporated the R1226A, a mutation that has not been tested in the context of transcriptional control. Relative to WT AsCasl2a, the AsCasl2a R1226A mutant protein, described as a nickase in its original characterization (Yamano et al. 2016), is -100- 1,000 fold slower in cleaving the non-target DNA strand and - 10,000-fold slower in cleaving the target DNA strand in vitro (Cofsky et al. 2020). Consistent with nicking-induced stabilization, AsCasl2a R1226A indeed binds DNA more strongly in vitro than fully DNase- inactivated D908A variant (Cofsky et al. 2020). We expect the R1226A mutation to both disfavor R-loop reversal and slow progression to double- stranded break (FIG. 2A; see Discussion). We hypothesize that, by trapping the complex in a nicked DNA intermediate, the R1226A mutation would prolong chromatin occupancy and thus time available for the KRAB domain to recruit transcriptional repressive complexes.

[00107] To test the impact of R1226A on CRISPRi activity, we replaced the DNase- inactivating D908A in denAsCas 12a- KRAB with R1226A, and hereafter refer to this Casl2a variant as multiAsCasl2a (multiplexed transcriptional interference, i.e.

R1226A/E174R/S542R/K548R). To test their CRISPRi performance at different protein doses, we lentivirally transduced denAsCas 12a- KRAB and multiAsCas 12a- KRAB constructs at MOI = ~1 vs. MOI = ~5 in K562 cells and sorted for stably expressing BFP-positive cell population. Using lentivirally delivered crRNAs targeting CD81 and CD55 as single crRNAs and as part of 3-plex crRNAs, we compared the CRISPRi performance of multiAsCas 12a- KRAB vs. denAsCas 12a- KRAB across different combinations of protein MOI and crRNA MOIs. Across a panel of single and 3-plex crRNA constructs, we found that multiAsCas 12a- KRAB consistently achieves robust CRISPRi with less sensitivity to low protein MOI, and with especially large improvements over denAsCas 12a- KRAB in the setting of low crRNA MOI (FIG. 2B and FIG. 16) and especially for 3-plex crRNAs (FIG. 2C and FIG. 17). As a notable example, a 3-plex crRNA (crCD81-l_crCD151-3_crCD55-4) that is virtually inactive for CD81 knockdown by denAsCas 12a- KRAB even in the setting of high protein MOI ~5 shows >95% median CD81 expression knockdown by multiAsCas 12a- KRAB. This same 3-plex crRNA construct shows double knockdown of CD55 and CD81 in only 14.3% of single cells for denAsCas 12a- KRAB vs. 76.7% for multiAsCas 12a- KRAB (FIG. 2D). Similarly, multiAsCas 12a- KRAB is able to rescue the CRISPRi activity of single and 3-plex crRNA constructs targeting CD151 that are otherwise completely inactive when used with denAsCas 12a- KRAB (FIG. 2E). multiAsCas 12a- KRAB CRISPRi activity shows generally minimal or no off-target effects on the transcriptome as evaluated by bulk RNA-seq (FIG. 18).

[00108] As the AsCasl2a R1226A mutant is known to cut both DNA strands slowly in vitro (Cofsky et al. 2020), we characterized the impact on DNA sequence of long-term constitutive targeting of multiAsCasl2a-KRAB to genomic sites in K562 cells. Using K562 cells lentivirally engineered with multiAsCas 12a- KRAB or denAsCas 12a- KRAB at protein MOI = -5, we tested for the DNA sequence alterations at CRISPRi target sites near the CD55 and CD81 TSS's after 20 days of constitutive ribonucleoprotein expression. Any DNA sequence alterations are expected to accumulate over this duration, which is representative of the upper end of duration for typical CRISPR functional genomics experiments. We reasoned that testing a highly effective CRISPRi crRNA (crCD55-4) would represent an experimental condition of high target occupancy over a long time period, enabling us to estimate the upper end of indel formation. In this experiment, we observed indel frequencies of 7.9% by multiAsCasl2a- KRAB, 0.1% by denAsCasl2a-KRAB, and 97.9% by a fully DNase-active construct, opAsCasl2a (Gier et al. 2020) (FIG. 2F). For crCD81-l, a crRNA with an intermediate level of CRISPRi activity more representative of most crRNAs we have tested, we observed indel frequencies of 2.9% by multiAsCasl2a-KRAB, 0.87% by denAsCasl2a, and 76.5% by opAsCasl2a (FIG. 2F). If deletions were the sole driver of target gene knockdown, even if assuming a deletion of any size generates a complete null CD81 allele, in this triploid region of the K562 genome we would expect -89.5% of cells to harbor zero indcls and retain full expression level, -10.1% of cells to harbor a deletion of any size in one DNA copy and thus retain -67% of CD81 expression level, -0.38% of cells to inactivate two DNA copies, and virtually no cells to inactivate all 3 DNA copies. The expected change in median CD81 expression in the cell population under this adversarial assumption of abolishing expression in cis by any sized deletion would be -3.7% (see Methods). This expectation is wholly inconsistent with the magnitudes of median CD81 expression knockdown by multiAsCasl2a-KRAB, including up to -95% knockdown in excess of denAsCasl2a-KRAB for crCD81-l_crCD151- 3_crCD55-4 (FIG. 2C, far right subpanel). This is the first of multiple lines of evidence, further addressed in subsequent sections, demonstrating that the magnitude of target gene knockdown by multiAsCasl2a-KRAB is far from being accounted for by DNA sequence alterations alone. [00109] multiAsCas!2a-KRAB enables CRISPRi using higher-order multiplexed crRNA arrays delivered by lentiviral transduction. To test the performance of multiAsCasl2a-KRAB in targeting >3 genomic sites per cell for CRISPRi, we designed a lentiviral system for expressing higher-order multiplexed crRNA arrays, keeping the overall U6 promoter and CROP-seq vector design with a 3' direct repeat (Gier et al. 2020). To minimize the possibility of lentiviral recombination, this system (FIG. 3A) uses a unique direct repeat variant at each position of the array, selected from a set of direct repeat variants previously engineered and screened for strong activity in the setting of enAsCasl2a DNA cutting in human cells (DeWeirdt et al. 2020). Using this lentiviral expression system, we assembled a panel of 13 distinct crRNA constructs (7 single-plex, two 3-plex, two 4-plex, two 5-plex, and two 6-plex), with the higher-order crRNA arrays assembled from individually active spacers targeting the TSS's of CD55, CD81, B2M and KIT (FIG. 19). For this panel of 13 crRNA constructs, we compared the CRISPRi activities of denAsCasl2a-KRAB, multiAsCasl2a-KRAB, and multiAsCasl2a (no KRAB) as a negative control for the impact of the KRAB domain. For a subset of crRNA constructs we also added enAsCasl2a-KRAB (DNase fully active) to test the effect of fully active DNA cutting in conjunction with the KRAB domain in target gene knockdown. For the remainder of the study we use piggyBac transposition to constitutively express all fusion protein constructs, which yields results similar to that obtained from high MOI (-5) lentiviral delivery of protein constructs and avoids day-to-day variations in lentiviral titers. Each piggyBac-delivered construct is expressed in K562 cells at very similar protein levels as measured by western blot (FIG. 12) and routine flow cytometry monitoring of the P2A-BFP fluorescence signal (FIG. 20).

[00110] To summarize results of the full panel of crRNA constructs, multiAsCas 12a- KRAB substantially outperforms denAsCas 12a- KRAB in CRISPRi activity for 7 out of 7 constructs tested for CD81 knockdown (FIG. 3B); 5 out of 6 constructs tested for B2M knockdown (FIG. 19); and 6 out of 6 constructs tested for KIT knockdown (FIG. 3C). For CD55 (FIG. 19), multiCas 12a- KRAB substantially outperforms denAsCas 12a- KRAB for the single-plex crCD55-5 (weaker spacer), and performs either the same as or marginally better than denAsCas 12a- KRAB for all 7 constructs containing crCD55-4 (strongest spacer). These overall results are exemplified by a 6-plex crRNA (crCD55-4_crB2M-l_crB2M-3_crKIT-2_crKIT- 3_crCD81-l), for which multiAsCas 12a- KRAB substantially outperforms denAsCas 12a- KRAB in knockdown of B2M, KIT, and CD81, and marginally so for CD55 (FIG. 3B). Similarly superior CRISPRi performance by multiAsCas 12a- KRAB over denAsCas 12a- KRAB was also observed when using up to 6-plex crRNA arrays in a different cell type, C4-2B prostate cancer cells (FIG. 13).

[00111 ] For all crRNA constructs tested, multiAsCas 12a alone shows much lower impact on target gene expression than multiAsCas 12a- KRAB, demonstrating that the large improvements in gene knockdown by multiAsCas 12a- KRAB depends on the KRAB domain. However, for some target genes, such as KIT, partial knockdown can be observed for multiAsCasl2a alone. Such gene knockdown may be due to 1) direct obstruction of the transcriptional machinery, or 2) deletion of DNA sequences crucial for transcription via doublestranded break formation and repair. To test distinguish these possibilities, we quantified indels generated by the panel of fusion proteins using a 6-plex crRNA array (crCD55-4_crB2M- l_crB2M-3_crKIT-2_crKIT-3_crCD81-l) containing two crRNAs targeting opposite strands at sites 95bp apart near- the KIT TSS (FIG. 3D and FIG. 21, same 6-plex crRNA construct as included in FIG. 3C). This genomic distance between two crRNA binding sites is known to optimally facilitate deletions of the intervening region by DNA cutting Cas proteins (Joberty et al. 2020), thus represents an upper estimate of the frequencies of deleting intervening regions of multiple target sites in cis. The maximum indel frequencies measured for any given base were only 3%-5.4% for multiAsCasl2a and 1.2%-3.7% for multiAsCasl2a-KRAB at each individual crRNA binding site (FIG. 3D, region A and region C). In the intervening region between the two crRNA binding sites, both multiAsCasl2a and multiAsCasl2a-KRAB generated only a maximum of 0.2% indels (FIG. 3D, region B). For comparison, fully DNase-active enAsCasl2a-KRAB generates up to -94% indels at each individual crRNA binding site and up to 7.6% in the intervening region (FIG. 3D). Based on these measured indel frequencies and known triploidy of this K562 genomic region (Zhou et al. 2019), we simulated the expected single-cell gene expression distributions due to effects purely arising from the maximal observed frequencies of deletions of any size in the PCR amplicon (FIG. 21). Under this purely deletional assumption, we calculated an upper estimate of expected -1.8% median KIT expression knockdown by multiAsCasl2a-KRAB, far lower than the observed -90.4% median knockdown, which is -44.4% in excess of the observed for denAsCasl2a-KRAB (FIG. 21). These results demonstrate that target gene knockdown by multiAsCasl2a-KRAB is largely attributable to non-genetic perturbation of transcription. For multiAsCasl2a (without KRAB), the upper estimate of expected knockdown under the purely deletional assumption is -2.5%, vs. -67.7% observed (FIG. 21). In the absence of the KRAB domain, this observed knockdown likely reflects direct obstruction of the transcriptional machinery, especially by the crKIT-2 target site downstream of TSS. Similar trends were obtained for single-site targeting using crKIT-2 encoded within a 4-plex crRNA array (FIG. 22). We conclude that marginal effects of indels to target gene knockdown by multiAsCasl2a-KRAB do not affect interpretations in most functional genomics applications.

[00112] At the single cell level, multiAsCasl2a-KRAB consistently outperforms denAsCasl2a-KRAB in the fraction of single cells with successful double knockdown (FIG. 3E-F and FIG. 23) and triple knockdown (FIG. 3G and FIG. 23) of target genes using higher- order crRNA arrays. To further explore the performance of crRNA arrays at higher extremes of multiplexing, we constructed 8-plex and 10-plex constructs assembled using individually active spacers. In these 8-plex and 10-plex arrays, spacers encoded in multiple positions within the array maintain robust CRISPRi activity (i.e. for CD55, KIT and B2M, Fig 3H). crCD81-l encoded at the 3' most position shows progressive diminishment in CRISPRi activity with further multiplexing at 8-plex and 10-plex (FIG. 3H). These results indicate that 8-plex and 10- plex crRNA arrays at least partially support robust CRISPRi activity for most spacers within these arrays. The diminishment in CRISPRi activity for crCD81-l is not clearly attributable solely to the length of the crRNA array per sc but may also involve contributions from deficiencies driven by local sequence context and further exacerbated by reduction of available fusion protein to bind each individual crRNA upon further multiplexing. We also observed that a specific 6-plex crRNA construct (crCD81-l_crB2M-l_crB2M-3_crKIT-2_crKIT-3_crCD55- 4, 6-plex #2 in FIG. 3F) fails to knockdown B2M while achieving robust CRISPRi of the other target genes. However, the same combination of spacers in a slightly different 6-plex arrangement (crCD55-4_crB2M-l_crB2M-3_crKIT-2_crKIT-3_crCD81-l) and also in 8-plex and 10-plex embodiments achieve decent B2M knockdown. These results suggest the existence of still unknown local sequence context influences on CRISPRi activity of specific spacers within crRNA arrays that can be separate from influences of array length.

[00113] multiAsCasl2a-KRAB outperforms denCas!2a-KRAB in pooled CRISPRi screens and shows TSS-proximal activity patterns and overall potency similar to dCas9- KRAB. Given the success of multiAsCasl2a-KRAB in individual well-based assays using lentivirally delivered crRNAs, we next evaluated its performance in the context of high- throughput pooled screens using sequencing to quantify the activities of each crRNA construct within a pooled library. We designed a library, referred to as Library 1, aimed at extracting patterns for Casl2a CRISPRi activity with respect to proximity to the TSS using cell fitness as a readout. Library 1 contains 77,387 single crRNA lentiviral constructs tiling all predicted canonical TTTV PAM sites and non-canonical PAM’s (recognizable by enAsCasl2a (Kleinstiver et al. 2019)) in the -50bp to +300bp region around the TSS's of 559 common essential genes with K562 cell fitness defects in prior genome-wide dCas9-KRAB screens (Horlbeck et al. 2016a). The library also includes two types of negative controls: 1) 524 crRNAs targeting intergenic regions away from predicted regulatory elements across all human cell lines based on ENCODE chromatin accessibility data, and 2) 445 non-targeting crRNAs that do not map to the human genome (FIG. 24).

[00114] Using K562 cells constitutively expressing multiAsCasl2a-KRAB or denAsCasl2a-KRAB, we transduced cells with this TSS tiling crRNA library (MOI = 0.15), collected a sample at the start of the screen and then carried out the cell fitness screen for -8 total cell population doublings in replicate. Genomic DNA was extracted from each sample and the relative abundance of each crRNA in the cell population was measured by sequencing. In this assay, cell fitness defects due to CRISPRi knockdown of target essential genes is reflected in depletions of crRNA sequencing read abundances over the duration of the screen. Using read abundances normalized to the medians of negative control crRNAs, we calculate a cell fitness score for each crRNA construct that quantifies the fractional fitness defect per cell population doubling (defined as y in (Kampmann et al. 2013)). Concordance between cell fitness scores of screen replicates is high for multiAsCasl2a-KRAB (R = 0.71) and much lower for denAsCasl2a-KRAB (R = 0.32), the latter due to much lower signal-to-background ratio (FIG. 25). The cell fitness score distributions are virtually indistinguishable between the intergenic targeting negative controls and the non-targeting negative controls (FIG. 26), indicating no appreciable non-specific genotoxicity from multiAsCasl2a-KRAB single-site targeting. Among the 3,326 crRNA's targeting canonical TTTV PAM's, 24.5% vs. 17.5% showed a fitness defect in multiAsCasl2a-KRAB vs. denAsCasl2a-KRAB, respectively (using the 5th percentile of intergenic negative controls as a threshold), with the magnitude of effect for each crRNA overall stronger for multiAsCasl2a-KRAB (FIG. 4B). In contrast, crRNAs targeting non-canonical PAMs show fitness scores less clearly distinguishable from the negative control distribution (FIG. 4C). We compared our observed cell fitness scores to on-target activity predictions by CRISPick, the state-of-the-art crRNA activity prediction algorithm trained on enAsCasl2a DNase screening data (DeWeirdt et al. 2021; Kim et al. 2018). Because CRISPick on-target predictions arc already tightly associated with whether a crRNA targets a TTTV or non- canonical PAM, we analyzed the predictive power of CRISPick within each of these two PAM categories separately (FIG. 4C). Within each PAM category, CRISPick on-target prediction scores weakly correlates with the observed cell fitness score (R = -0.18 for TTTV PAM and R = -0.1 for non-canonical PAMs), indicating significant sources of crRNA CRISPRi activity variation beyond what is modeled by CRISPick.

[00115] Previous studies using dCas9-KRAB have identified a strong association between CRISPRi activity and genomic proximity of the crRNA binding site to the TSS (Gilbert et al. 2014; Nunez et al. 2021). To facilitate direct comparison of our current multiAsCasl2a- KRAB and denCasl2a-KRAB TSS tiling analysis with prior dCas9-KRAB data, we focused on crRNAs targeting TTTV PAM's near the TSS's of 240 essential genes for which dCas9-KRAB tiling data is available (Nunez et al. 2021). The average cell fitness scores of these crRNAs at each genomic position relative to the TSS reveal a remarkably similar- bimodal pattern in CRISPRi activity as that obtained by dCas9-KRAB sgRNA's targeting NGG PAM's in the same genomic windows (FIG. 4D). The weakened activity centered around +125bp to +150bp region is consistent with hindrance by a well-positioned nucleosome (Nunez et al. 2021; Horlbeck et al. 2016b). multiAsCasl2a-KRAB shows similar magnitudes of averaged cell fitness scores as dCas9-KRAB in this meta-TSS analysis, whereas denCasl2a-KRAB is substantially weaker than both at all positions relative to the TSS (FIG. 4D). As another benchmark of CRISPRi activity across the 583 essential TSS's targeted in the screen, we compared the average CRISPRi activity of the top 3 best performing crRNAs/sgRNA for each TSS across the different proteins. The top 3 sgRNA's per TSS for dCas9-KRAB are taken from a prior genome-wide screen that used 10 sgRNAs per TSS, which were pre-selected based on bioinformatic prediction of strong sgRNA activity (Horlbeck et al. 2016a). In summary, large scale functional genomics experiments, which simultaneously examine CRISPRi activity across many crRNAs and genes, demonstrate multiAsCasl2a-KRAB outperforms denCasl2a-KRAB (FIG. 4E) and also that multiAsCasl2a-KRAB performs favorably when compared to dCas9-KRAB, though there is large variability across TSS's (FIG. 4E).

[00116] multiAsCas!2a-KRAB enables pooled sequencing screens using 6-plex crRNA arrays, despite positional biases in CRISPRi activity. To evaluate the performance of multiAsCasl2a-KRAB in pooled sequencing screens using multiplexed crRNA constructs, we constructed a library consisting of 6-plex crRNAs, as this length is supported by the upper end of commercially available pooled oligo synthesis. We refer to this 6-plex library as Library 2, which includes Sublibrary A (described in this section) and Sublibrary B (described in the next section). Sublibrary A was designed to contain 84,275 6-plex constructs for evaluating CRISPRi activity at each of the 6 positions in the array in a K562 cell fitness screen (FIG. 4E and FIG. 24). Each 6-plex construct has one of the 6 positions designated as the "test" position, which can encode either 1) a spacer targeting one of the top 50 essential gene TSS's (ranked based on prior dCas9-KRAB screen data (Nunez et al. 2021)), or 2) an intergenic negative control (FIG. 4E). The remaining 5 positions in the array are designated as "context" positions that encode negative control spacers drawn from a separate set of 30 negative control spacers (FIG. 4E). The motivation for this library design was to enable sampling multiple sets of context spacers for a given test position.

[00117] The entirety of Library 2 was used in a cell fitness screen conducted as described in the previous section but over -13.5 total cell population doublings in K562 cells stably expressing multiAsCasl2a-KRAB. The relative DNA abundances of the crRNA arrays in each sample was quantified by sequencing the crRNA array (see methods). As Library 2 was designed and cloned prior to the completion of the Library 1 screen, the majority of Library 2 contains constructs encoding for spacers in the test position that in hindsight do not produce strong phenotypes as single crRNAs in the Library 1 screen. Thus, we focused our analysis on 1) 2,987 6-plex crRNA arrays that encode in the test position one of 123 spacers with empirically strong cell fitness scores as single crRNAs in the Library 1 screen, and 2) 12,029 6- plex crRNA arrays that encode in the test position one of 506 negative control spacers (FIG. 24). We calculated the average cell fitness scores from the top 3 context constructs ranked by cell fitness score for a given test position spacer, applying this calculation equally to essential TSS-targeting test position spacers and negative control test position spacers (FIG. 4F). The top 3 context-averaged cell fitness scores for essential TSS-targeting spacer are clearly distinguishable from the negative control distributions at each test position in the 6-plex array (FIG. 4G), albeit with weaker magnitudes than for the same spacer encoded as individual single crRNA in the Library 1 screen (FIG. 27). We used the 5th percentile of the intergenic negative control cell fitness score distribution in each test position as a threshold for calling whether the test position spacer shows successful CRISPRi activity. Using this threshold, we quantified the % recall, by the 6-plex arrays, of empirically active single crRNA spacers from the Library 1 screen (FIG. 4F). We observed an aggregate recall of 64% across all test positions, with better recall from the 1st (86%) and 5th (88%) test positions, and lower recall from the 6th test position (48%) (FIG. 4F). As these positional biases are not a monotonically decreasing function of distance from the U6 promoter, they are inconsistent with any potential abortive RNA Pol III transcription of the pre-crRNA being the sole driver of these biases. As each position in the array is assigned a unique direct repeat variant that is held constant across all constructs in this analysis, it is possible these apparent positional effects may reflect contributions from unknown properties intrinsic to the direct repeat variant sequences. As we have done, redundantly sampling the same combination of spacers encoded in different orders within arrays can reduce false negative results. We conclude that multiAsCasl2a-KRAB enables pooled sequencing screens using 6-plex crRNA arrays.

[00118] multiAsCas!2a-KRAB enables discovery and higher-order combinatorial perturbations of cis-regulatory elements. The human genome contains -500,000 predicted enhancers, but only a small minority have been functionally tested by perturbations. Previous studies have shown that dCas9 CRISPRi can outperform Cas9 DNA cutting in perturbing enhancer function in pooled screens (Ren et al. 2021; Tycko et al. 2019), likely due to the broader genomic window DNA that is perturbed by formation of repressive chromatin versus indels generated by individual guide RNAs. To our knowledge, no study has reported enhancer perturbation by CRISPRi using Casl2a. We confirmed that multiAsCasl2a-KRAB targeting using single crRNAs can effectively perturb a known enhancer of the HBG gene, HS2 (FIG. 5A), with knockdown of HBG mRNA expression in K562 cells comparable to the effect of dCas9-KRAB targeting HS2 (Li et al. 2020).

[00119] We next aimed to use multiAsCasl2a-KRAB to discover previously uncharacterized enhancers using the CD55 locus in K562 cells as a myeloid cell model. CD55 encodes for decay-accelerating factor, a cell surface protein that inhibits the activation of complement and is expressed in most human cell types (Dho et al. 2018). CD55 function in the myeloid lineage is particularly relevant in multiple disease states, including paroxysmal nocturnal hemoglobinuria (Hillmen et al. 2004) and malaria (Egan et al. 2015; Shaky a et al. 2021). To our knowledge, no known enhancers in myeloid cells have been identified for CD55. In K562 cells, several DNase hypersensitive sites (DHS) bearing histone 3 lysine 27 (H3K27Ac), a modification associated with active enhancers, reside near CD55 (FIG. 5B). The activity-by-contact (ABC) enhancer prediction algorithm (Fulco et al. 2019) predicts 4 of these DHSs (R1-R4) as candidate enhancers (FIG. 5B). While R1-R3 reside in a region between 3kb- 1 Ikb upstream of the CD55 promoter, R4 sits in an intronic region of the Clorfl 16 gene, ~297kb away from the CD55 promoter (FIG. 5B). To conduct a focused screen of the DHSs within this general region for enhancers that regulate CD55, we designed a total of 21 4-plex crRNAs (encompassing 88 individual spacers) targeting 11 regions bearing varying levels of DNase hypersensitivity and H3K27Ac (R1-R4 predicted by ABC; R5-R11 picked manually), plus a negative control region (R12) devoid of DHS and H3K27Ac. The regions (except RIO and R12) are each independently targeted by two 4-plex crRNAs. Each 4-plex crRNA was lentivirally transduced into K562 cells expressing multiAsCasl2a-KRAB, followed by flow cytometry readout of CD55 expression. We found that the ABC-predicted R1-R4 show ~50%- 75% reduction in CD55 expression upon multiAsCasl2a CRISPRi targeting, whereas no decrease in CD55 expression is observed for R5-R12. For each of the functionally validated Rl- R4 enhancers, the two 4-plcx crRNA arrays that target each enhancer show quantitatively similar levels of CD55 knockdown (FIG. 5B), indicating each array contains an active 4-plex or lower-order active combination of spacers. This consistency in the magnitude of CD55 expression knockdown likely reflects the magnitude of true enhancer impact on gene transcription, rather than technical peculiarities of individual spacer activities, which might be more unpredictably variable and labor-intensive to test if encoded as single-plex perturbations. In contrast to multiAsCasl2a-KRAB, using opAsCasl2a to target R1-R4 for DNA cutting using the same 4-plex crRNAs elicits very little or no CD55 expression knockdown, despite potent knockdown by a positive control crRNA targeting a coding exon (FIG. 5C). This demonstrates a key advantage of multiAsCasl2a-KRAB over state-of-the-art Casl2a DNA cutting tools for perturbing enhancer function, even in the setting of multiple crRNA target sites within the same enhancer. To our knowledge, R1-R4 are the first functionally demonstrated enhancers for CD55 in a myeloid cell type, in addition to another CD55 enhancer that was recently reported in a B- cell model (Cheng et al. 2022).

[00120] To further test the utility of multiAsCasl2a-KRAB in studies of enhancer function, we used the MYC locus as a model. MYC is an essential gene in most proliferative cells when perturbed by CRISPRi, enabling the use of cell fitness as a readout in pooled screens to identify genomic elements that regulate MYC expression. Prior studies using CRISPRi pooled screens in K562 cells have shown that MYC expression is proportional to cell fitness and is regulated by several enhancers identified by both screens using cell fitness (Fulco et al. 2016) and mRNA expression (Reilly et al. 2021) readouts. A recent study found that pairwise dCas9- KRAB perturbations of these enhancers showed stronger phenotypes than perturbing single enhancers (Lin et al. 2022). In that study, a single-step large scale pooled screen was used to test 295 x 295 = 8,7025 pairs of guide RNAs targeting known MYC enhancers in a single step. To our knowledge, no study has reported the phenotypic impact of 3-plex or higher-order perturbations of regulatory elements at the MYC locus.

[00121] We used multiAsCas 12a-KRAB to dissect higher-order combinatorial cis- regulation at the MYC locus. To avoid testing intractably numerous higher-order combinations of crRNA spacers that are largely uninformative due to the inclusion of weak or inactive crRNA spacers, we opted to pre-screen for a small group of active 3-plex crRNA combinations that can be assembled into higher-order combinations in a subsequent step. We used multiAsCas 12a- KRAB to test four 3-plcx crRNA constructs targeting combinations of MYC cis-rcgulatory elements (3 crRNAs for promoter and 3 crRNAs for each of 3 known enhancers, el, e2 and e3) in a well-based cell competition assay (FIG. 6D). We found that these four 3-plex crRNAs induce varying degrees of cell fitness defect as a proxy of MYC expression knockdown. This pre-nomination step indicates that each construct contains some 3-plex or lower-order spacer combinations that exhibit CRISPRi activity (FIG. 6D). For comparison, we included denAsCasl2a-KRAB, multiAsCas 12a, enAsCasl2a-KRAB and opAsCasl2a as controls, which showed consistent relative activities in MYC knockdown phenotype in further support of our conclusions (based on FIG. 2 and FIG. 3) regarding multiAsCasl2a-KRAB's superior CRISPRi potency and the minimal or undetectable impact of its DNA cutting activity (FIG. 6D).

[00122] We then in silico assembled these 12 nominated spacers and 3 intergenic negative control spacers into Library 2 Sublibrary B, consisting of 6,3706-plex permutations encoded as 6-plex crRNA arrays (FIG. 6E). These 6-plex crRNA arrays each target up to 4 cis- regulatory elements (promoter + 3 enhancers) with up to 3 spacers per element. Negative control spacers fill in the remaining positions in arrays that are not fully filled by targeting spacers. This Sublibrary B was included as part of the cell fitness screen for the entirety of Library 2, as described in the previous section. Among 1,823 6-plex arrays with sufficient read coverage for analysis, we grouped them into 16 categories, based on whether it encodes at least 1 spacer targeting the promoter, and/or at least 1 spacer targeting each of the 3 enhancers (FIG. 24). Examining the distributions of cell fitness scores within each category (FIG. 6F). 6-plex crRNA arrays targeting only el, e2, or e3 alone showed a modest cell fitness defect, with e2 having the strongest effect of all 3 enhancers (FIG. 6D, left panel). Co-targ eting each additional enhancer increased the magnitude of fitness defect, such that the crRNA arrays co-targeting el/e2/e3 shows the strongest fitness defect (FIG. 6D, left panel). Co-targeting the promoter together with any combination of enhancers showed increased cell fitness defect over targeting the promoter alone (FIG. 6D, right panel). These results suggest that when targeting subsets of cis-regulatory elements in a locus by CRISPRi components, other cis-regulatory elements can compete with the CRISPRi repression to sustain partial levels of gene transcription. Such effects may reflect how cis-regulatory elements combinatorially respond to endogenous repressive cues in the natural regulation of MYC gene transcription. Integrating the activities of multiple cis- regulatory elements may enable the cell to fine-tune the expression of this key regulator of cell growth in a physiologically appropriate manner.

Discussion

[00123] In this study, we engineered multiAsCasl2a-KRAB as a new platform for higher-order combinatorial CRISPRi perturbations of gene transcription and enhancer function. The enhanced CRISPRi potency of multiAsCasl2a-KRAB is more robust to lower effective concentrations of ribonucleoprotein, critically enabling high-throughput pooled screening applications conducted at single-copy integrations of crRNA expression. We propose that the improved CRISPRi activity of multiAsCasl2a-KRAB emerges from prolonged chromatin occupancy due to DNA nicking. This strategy is conceptually distinct from prior protein engineering approaches to improving Casl2a function in mammalian cells, which focused on substituting for positively charged amino acid residues near the proteimDNA interface (Kleinstiver et al. 2019; Guo et al. 2022), or used directed evolution focused on optimizing DNA cleavage (Zhang et al. 2021). In the absence of nicking, R-loop reversal occurs by invasion of the crRNA:target strand DNA duplex by the non-target strand to displace the crRNA, in a process analogous to toehold-mediated nucleic acid strand displacement (Srinivas et al. 2013). Severing the non-target strand effectively destroys the toehold and is expected to massively decrease the rate of DNA release (Srinivas et al. 2013) (FIG. 2A). Severing the non- target strand likely contributes to stabilizing the DNA-bound complex by imparting an entropic penalty to strand invasion for R-loop reversal. This mechanistic model can also explain previous observations of cutting-dependent complex stabilization (Cofsky et al. 2020; Knott et al. 2019; Singh et al. 2018). In invoking well-established principles of nucleic acid hybridization, this model suggests that favoring nicked DNA intermediates may be a generalizable strategy for improving the activities of other Cas enzymes in chromatin targeting. Another potential explanation for multiAsCasl2a's enhanced CRISPRi activity may be the formation of proteimDNA contacts after non-target-strand nicking that stabilize the complex (Naqvi et al., 2022). Separately, DNA nicking is expected to relax local supercoiling, which might inhibit nearby transcription (Baranello et al. 2012).

[00124] The multiAsCasl2a-KRAB platform enables new solutions to addressing major challenges in combinatorial genetics. With increased crRNA multiplexing beyond >3-plex combinations, the combinatorial space rapidly explodes in size, rendering exhaustive testing of all combinations cumbersome or infeasible. However, testing a single higher-order N-plcx combination also indirectly tests all or many of its constituent lower-order combinations, for up to a total of 2 ^N combinations. Thus, increases in multiplexing capability potentially yield exponential increases in search efficiency using the general concept of group testing (Dorfman 1943; Du 1993). In group testing (FIG. 7), a primary screen is conducted on grouped subjects (e.g. a multiplexed array of crRNA constructs) to reduce the costs otherwise incurred by individually testing all subjects (e.g. an individual crRNA). Our screening for CD55 enhancers instantiates this approach by testing 22 4-plex crRNA arrays targeting 12 candidate regions, therefore indirectly testing 22 x 2 ⁴ = 352 crRNA combinations in a cost-effective experiment using flow cytometry to assay just 22 wells in a plate (FIG. 5B-C). For this experimental objective, the grouped hits can be biologically interpreted without further testing (FIG. 7). For other objectives, such as the combinatorial analysis of cis-regulation at the MYC locus (FIG. 6D-F), grouped hits can be followed by a focused secondary screen as needed (FIG. 7). These results together demonstrate that the group testing framework can be used flexibly for individual well-based assays and/or pooled screening readouts. For pooled screens with sequencing readouts, the ability to deterministically program only specific higher-order combinations of compact Casl2a crRNAs by oligo synthesis for the initial screen is crucial for group testing. In contrast, cloning combinatorial guide libraries by a multiplicative and stochastic approach (Zhou et al. 2020) requires testing all combinations at the onset, and thus are incompatible with group testing. The application of group testing can significantly compress the size of crRNA libraries screens to facilitate functional genomics screens in more complex biological systems limited by assayable cell numbers, such as in vivo and organoid models. Group testing may also be combined with compressed sensing algorithms to further enable exploring genetic interactions in high-dimensional phenotypic spaces (Yao et al. 2023).

[00125] A parameter in group testing is the extent of potential signal dilution relative to individual testing. For Casl2a perturbations, this can arise in the form of 1) low doses of ribonucleoprotein due to limitations in delivery, such as crRNA expression from single-copy integrations in pooled sequencing screens, or 2) increased multiplexing, which effectively dilutes the concentration of functional Casl2a protein available to bind each individual crRNA. Despite some signal dilution in the stringent setting of pooled screens using 6-plex crRNAs expressed from single-copy integrants, multiAsCasl2a-KRAB demonstrates sufficient potency to yield new biological insights into combinatorial cis -regulation at the MYC locus using pooled screening of 6-plex crRNA arrays. While we have focused our optimizations to meet the stringent single-copy crRNA integration requirement of pooled screening formats, multiAsCasl2a also significantly lowers technical barriers to higher-order combinatorial perturbations in array-based screening, which is compatible with more diverse phenotypic readouts and has recently improved significantly in throughput (Yin et al. 2022). The assay format will likely influence the deliverable dose of synthetic components and thus the absolute upper limit of multiplexing for effective CRISPRi using multiAsCasl2a-KRAB, which currently remains uncertain. Among the spacers examined in the largest crRNA array we tested (10-plex), 3 spacers performed the same as each does in shorter arrays, while one showed substantially diminished CRISPRi activity (FIG. 3F).

[00126] While we have focused on CRISPRi applications using the KRAB domain in the present study, the discovery and engineering of effector domains for chromatin perturbations by CRISPR-Cas is a rapidly evolving area of research. Recent advances include new repressive effectors (Alerasool et al. 2020; Mukund et al. 2023; Replogle et al. 2022b; DelRosso et al. 2023), activation effectors (Alerasool et al. 2022; Mukund et al. 2023; DelRosso et al. 2023), and combination effectors for epigenetic memory (Nunez et al. 2021; Van et al. 2021; Nakamura et al. 2021; Amabile et al. 2016). MultiAsCasl2a can be flexibly combined with these and other effector domains to enable group testing for many chromatin perturbation objectives. multiAsCasl2a and the group testing framework will enable tackling challenges in genome regulation and combinatorial genetics at previously intractable scales.

Methods

[00127] Plasmid Design and Construction. A detailed table of constructs generated in this study will be provided as a Supplemental File with all sequences. Constructs will be made available on Addgene. Cloning was performed by Gibson Assembly of PCR amplified or commercially synthesized gene fragments (from Integrated DNA Technologies or Twist Bioscience) using NEBuilder Hifi Master Mix (NEB Cat# E262), and final plasmids sequence- verified by Sanger sequencing of the open reading frame and/or commercial whole-plasmid sequencing service provided by Primordium.

[00128] Protein constructs components: The denAsCasl2a open reading frame was PCR amplified from pCAG-dcnAsCasl2a(E174R/S542R/K548R/D908A)-NLS(nuc)-3xHA- VPR (RTW776) (Addgene plasmid # 107943, from (Kleinstiver et al. 2019)). AsCasl2a variants described were generated by using the denAsCasl2a open reading frame as starting template and introducing the specific mutations encoded in overhangs on PCR primers that serve as junctions of Gibson assembly reactions. opAsCasl2a is from (Gier et al. 2020), available as Addgene plasmid # 149723, pRG232). 6xMyc-NLS was PCR amplified from pRG232. KRAB domain sequence from K0X1 was previously reported in (Gilbert et al. 2013). The lentiviral backbone for expressing Casl2a fusion protein constructs are expressed from an SFFV promoter adjacent to UCOE and is a gift from Marco Jost and Jonathan Weissman, derived from a plasmid available as Addgene 188765. XTEN80 linker sequence was taken from (Nunez et al. 2021) and was originally from (Schellenberger et al. 2009). For constructs used in piggyBac transposition, the open reading frame was cloned into a piggyBac vector backbone (Addgene #133568) and expressed from a CAG promoter. Super PiggyBac Transposase (PB210PA-1) was purchased from System Biosciences.

[00129] dAsCas 12a- KRAB x3 open reading frame sequence is from (Campa et al. 2019), in that study encoded within a construct referred to as SiT-ddCasl2a-[Repr], We generated SiT- ddCasl2a-[Repr] by introducing the DNase-inactivating E993A by PCR-based mutagenesis using SiT-Casl2a-[Repr] (Addgene #133568) as template. Using Gibson Assembly of PCR products, we inserted the resulting ddCasl2a-[Repr] open reading frame in-frame with P2A- BFP in a piggyBac vector (Addgene #133568) to enable direct comparison with other fusion protein constructs cloned in the same vector backbone (crRNA's are encoded on separate plasmids as described below).

[00130] Fusion protein constructs described in FIG. 15 were assembled by subcloning the protein-coding sequences of AsCasl2a and KRAB into a lentiviral expression vector using the In-Fusion HD Cloning system (TBUSA). AsCasl2a mutants were cloned by mutagenesis PCR on the complete wildtype AsCasl2a vector to generate the final lentiviral expression vector.

[00131] crRNA expression constructs: Unless otherwise specified, individual single and 3-plex As. crRNA constructs were cloned into the human U6 promoter-driven expression vector pRG212 (Addgene 149722, originally from (Gier et al. 2020)). Library 1, Library2, some 3-plex and all 4-plex, 5-plex, and 6-plex As. crRNA constructs were cloned into pCH67, which is derived from pRG212 by replacing the 3' DR with the variant DR8 (DcWcirdt ct al. 2021). For constructs cloned into pCH67, the specific As. DR variants were assigned to each position of the array as follows, in 5' to 3' order:

[00132] 3-plex: WT DR. DR1, DR3, DR8

[00133] 4-plex: WT DR, DR1, DR10, DR3, DR8

[00134] 5-plex: WT DR, DR1, DR16, DR10, DR3, DR8

[00135] 6-plex: WT DR. DR1, DR16, DR18, DR10, DR3, DR8

[00136] 8-plex: WT DR, DR1, DR16, DR.NSl, DR17, DR18, DR10, DR3, DR8

[00137] 10-plex: WT DR, DR1, DR16, DR_NS1, DR4, DR_NS2, DR17, DR18, DR10, DR3. DR8

[00138] Where the sequences of DR_NS 1 and 2 were based on combining hits from the variant DR screen from DeWeirdt et al., 2020. The sequences are DR_NS1: aattcctcctcttggaggt, and DR_NS2: aattcctcctataggaggt.

[00139] 1-plex, 3-plex, 8-plex, and 10-plex crRNA constructs were cloned by annealing complementary oligos, phosphorylation by T4 polynucleotide kinase (NEB M0201S), and ligated with T4 DNA ligase (NEB M0202) into BsmbI site of vector backbones. 4-plex, 5-plex and 6-plex crRNA arrays were ordered as double- stranded gene fragments and cloned into the BsmbI site of vector backbones by Gibson Assembly. [00140] Design of individual crRNAs. For cloning individual crRNA constructs targeting TSS's, CRISPick was used in the enAsCasl2a CRISPRi mode to design spacers targeting PAM's located within -50bp to +300bp region around the targeted TSS. We manually selected spacers from the CRISPick output by picking TTTV PAM-targeting spacers (except for crCD151-3, which targets a non-canonical GTTC PAM) with the highest On-Target Efficacy Scores and generally excluded any spacers with high off-target predictions. The same nontargeting spacer was used throughout the individual well-based experiments and was randomly generated and checked for absence of alignment to the human genome by BLAT (Kent 2002). [00141] The hgl9 genomic coordinates for MYC enhancers are: el chr8: 128910869- 128911521, e2 chr8: 128972341-128973219, and e3 chr8: 129057272-129057795. DNA sequences from those regions were downloaded from the UCSC Genome Browser and submitted to CRISPick. The top 3 spacers targeting TTTV PAM's for each enhancer were picked based on CRISPick On-target Efficacy Score, having no Tier I or Tier II Bin I predicted off-target sites, and proximity to the zenith of the ENCODE DNase hypersensitivity signal in K562 cells.

[00142] Cell culture, lentiviral production, lentiviral transduction. All cell lines were cultured at 37deg. C with 5% CO2 in tissue culture incubators. K562 and C4-2B cells were maintained in RPMI-1640 (Gibco cat# 22400121) containing 25 mM HEPES, 2mM L- glutamine, and supplemented with 10% FBS (VWR), 100 units/mL streptomycin, and 100 mg/mL penicillin. For pooled screens using K562 cells cultured in flasks in a shaking incubator, the culture media was supplemented with 0.1% Pluronic F-127 (Thermo Fisher P6866).

[00143] HEK 293T cells were cultured in media consisting of DMEM, high glucose (Gibco 11965084, containing 4.5g/mL glucose and 4mM L-glutamine) supplemented with 10% FBS (VWR) and lOOunits/mL streptomycin, lOOmg/mL penicillin. Adherent cells are routinely passaged and harvested by incubation with 0.25% Trypsin-EDTA (Thermo Fisher 25200056) at 37deg. C for 5-10min, followed by neutralization with media containing 10% FBS.

[00144] Unless otherwise specified below, lentiviral particles were produced by transfecting standard packaging vectors into HEK293T using TransIT-LTl Transfection Reagent (Minis, MIR2306). At <24 hours post-transfection culture media with exchanged with fresh media supplemented with ViralBoost (Alstem Bio, cat# VB 100) at 1:500 dilution. Viral supernatants were harvested -48-72 hours after transfection and filtered through a 0.45 mm PVDF syringe filter and either stored in 4deg. C for use within <2 weeks or stored in -80deg. C until use. Lentiviral infections included polybrene (8 mg/ml).

[00145] For experiments described in FIG. 15, lentivirus was produced by transfecting HEK293T cells with lentiviral vector, VSVG and psPAX2 helper plasmids using polyethylenimine. Media was changed ~6-8 h post transfection. Viral supernatant was collected every 12 h for 5 times and passed through 0.45 pm PVDF filters. Lentivirus was added to target cell lines with 8 pg/mL Polybrene and centrifuged at 650 x g for 25 min at room temperature. Media was replaced 15 h post infection. Antibiotics (1 pg/mL puromycin) was added 48 h post infection.

[00146] Antibody staining and flow cytometry. Antibodies used: CD55-APC (Biolegend 311312), CD81-PE (Biolegend 349506), B2M-APC (Biolegend 316311), KIT-PE (Biolegend 313204). Cells were stained with antibodies staining in 96-well plates, using 500g 5min at 4deg. C for centrifugation steps and decanting in between each step. Cells were washed once with 200ul with FACS Buffer (PBS with 1% BSA), then resuspended in 50ul of antibodies diluted at 1: 100 in FACS Buffer for 30min at 4deg. C. Then 150ul of FACS Buffer was added, followed by centrifugation and supernatant, then washed one more time with 200ul FACS buffer, followed by final resuspension in 200ul FACS Buffer for flow cytometry. For CRISPRi experiments, all data points shown in figures are events first gated for single cells based on FSC/SSC, then gated on GFP-positivity as a marker for cells successfully transduced with crRNA construct. Flow cytometry was performed on the Attune NxT instrument unless otherwise specified.

[00147] For cell fitness competition assays, the percentage of cells expressing the GFP marker encoded on the crRNA expression vector is quantified by flow cytometry. Iog2 fold change of % GFP-positive cells was calculated relative to day 2 (for experiments targeting the Rpa3 locus in FIG. 15) or day 6 (for experiments targeting the MYC locus in FIG. 6B). For experiments targeting the Rpa3 locus, flow cytometry was performed on the Guava Easycyte 10 HT instrument.

[00148] Indel analysis. 200K cells were collected on day 14 after crRNA transduction and genomic DNA was isolated using NucleoSpin Blood (Macherey-Nagel, Catalog no. 740951.50). Briefly, PCRs for loci of interest were run using Amplicon-EZ (Genewiz) partial IlluminaO adapters and amplicons were processed using NucleoSpin Gel and PCR Clean-up Kit (Macherey-Nagel, Catalog no. 740609.250). Paired end (2 x 250 bp) sequencing was completed at GENEWIZ (Azenta Life Sciences). Raw fastq files were obtained from GENEWIZ and aligned to reference sequences using CRISPResso2 (Clement et al. 2019) with the following modifications:

[00149] — quantification_window_size 12

[00150] — quantification_window_center -3

[00151] CRISPResso -fastq_rl Rl. fastq. gz — fastq_r2 R2.fastq.gz — amplicon_seq acccgtcttgtttgtcccacccttggtgacgcagagccccagcccagaccccgcccaaag cactcatttaactggtattgcggagccacga ggcttctgcttactgcaactcgctccggccgctgggcgtagctgcgactcggcggagtcc cggcggcgcgtccttgttctaacccggcgc gccatgaccgtcgcgcggccgagcgtgcccgcggcgctgcccctcctcggggagctgccc cggctgctgctgctggtgctgttgtgcct gccggccgtgtggggtgagtaggggcccggcggccggggaagcccctgggctgggtggga ggtccaagtcggtctctgaga -g actggtattgcggagccacgagg -wc -3 -w 12 (SEQ ID NO: 2) [00152]

[00153] For crRNA constructs in which the PAM is found on the opposite strand with respect to the amplicon sequence (in this case, CD81) the following modifications were included:

[00154] — quantification_window_size 20

[00155] — quantification_window_center - 18

[00156] CRISPResso — fastq_rl pCH45H-CD81-array_S5_L001_Rl_001.fastq.gz — fastq_r2 pCH45H-CD81-array_S5_L001_R2_001.fastq.gz — amplicon_seq ctgcttcgcggggacgaggggggggctcgcgggcgggactcctggcgccccgcccccatg agctcatcaagagccgccgcccctgg atggtggggcgggggcgcacactttgccggaggttgggggcgatccgcctcactctttcc ccagcccagctcactctccaatctgcggtc accacccgagaccttcctgggggtcgcgcctaaaaggagcgcagactcccgccgggatgg cccagaagctggggtgcgcgcaccctg gccgtccctgcctgggagccgatctccctctcctcacccagacacgttccagcggaggcc tcctcccagaagggctctggaggcctcgc aggagtggggatcccgcggttctgagttgg -p 3 -g gagaccttcctgggggtcgcgcc -wc -18 -w 20 (SEQ ID NO: 3)

[00157] Quantification diagrams were generated in R.

[00158] For analysis of dual cutting at the KIT TSS, briefly, DNA was isolated using QuickExtract DNA Solution (Lucigen) and amplicons were generated using 15 cycles of PCR to introduce Illumina sequencing primer binding sites and 0-8 staggered bases to ensure library diversity. After reaction clean-up using ExoSAP-IT kit (Thermo Fisher 78201), an additional 15 cycles of PCR was used to introduce unique dual indices and Illumina P5 and P7 adaptors. Libraries were pooled and purified by SPRIselect magnetic beads before paired-end sequencing using an Illumina MiSeq. Sequencing primer binding sites, unique dual indices (from Illumina TruSeq kits), P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 vl6.

[00159] Reads were analyzed using CRISPRessoBatch from CRISPResso2 (Clement et al. 2019) with the following modifications: wc -4 -w 15

[00160] CRISPRessoBatch — batch_settings batch2.batch — amplicon_seq aagagcaggggccagacgCCGCCGGGAAGAAGCGAGACCCGGGCGGGCGCGAGGGAGGGG AGGCGAGGAGGGGCGTGGCCGGCGCGCAGAGGGAGGGCGCTGGGAGGAGGGGCT GCTGCTCGCCGCTCGCGGCTCTGGGGGCTCGGCTTTGCCGCGCTCGCTGCACTTGGG CGAGAGCTGGAACGTGGACCAGAGCTCGGATCCCATCGCAGCTACCGCGATGAGA GGCGCTCGCGGCGCCTGGGATTTTCTCTGCGTTCTGCTCCTACTGCTTCGCGTCCAG ACAGGTGGGACACCGCGGCTGGCACCCCGACCGTGcgactactcggcgaagcctgtg -p 3 -g TCTGCGTTCTGCTCCTACTGCTT -wc -4 -w 15 (SEQ ID NO: 4)

[00161 ] For dual gRNA cutting, both guides were included in the batch analysis.The total number of insertions and deletions at each amplicon position were calculated and displayed using the effect_vector_combined.txt output.

[00162] Frequencies of nucleotide substitutions with multiAsCasl2a-KRAB targeting are negligible and indistinguishable from sequencing error (<4.5%) observed in unmodified K562 cells.

[00163] Pooled crRNA library design. For all crRNAs in Library 1 and Library 2: we excluded in the analysis spacers with the following off-target prediction criteria using CRISPick run in the CRISPRi setting: 1) off-target match = 'MAX' for any tier or bin, and 2) # Off-Target Tier I Match Bin I Matches > 1). The only crRNAs for which this filter was not applied are the non-targeting negative control spacers, which do not have an associated CRISPick output. All crRNA sequences were also filtered for the absence of BsmbI sites used for cloning and >3 consecutive T's, which mimic RNA Pol III termination signal.

Library 1 (single crRNA's) [00164] To design crRNA spacers targeting gene TSS's for Library 1, we used the -50bp to +300bp regions of TSS annotations derived from capped analysis of gene expression data and can include multiple TSS's per gene (Horlbeck et al. 2016a). We targeted the TSS's of 559 common essential genes from DepMap with the strongest cell fitness defects in K562 cells based on prior dCas9-KRAB CRISPRi screen (Horlbeck et al. 2016a). We used CRISPick with enAsCasl2a settings to target all possible PAM's (TTTV and non-canonical) in these TSS- proximal regions. Except for the criteria mentioned in the previous paragraph, no other exclusion criteria were applied. For the TSS-level analyses shown in FIG. 4D-E, each gene was assigned to a single TSS targeted by the crRNA with the strongest fitness score for that gene. [00165] Negative controls in Library 1 fall into two categories: 1) intergenic negative controls, and 2) non-targeting negative controls. Target sites for intergenic negative controls were picked by removing all regions in the hgl9 genome that are within lOkb of annotated ensembl genes (retrieved from biomaRt from https://grch37.ensembl.org) or within 3kb of any ENCODE DNase hypersensitive site (wgEncodcRcgDnascClustcrcdV3.bcd from http://hgdownload.cse.ucsc.edu/goldenpath/hgl9/encodeDCC/wgE ncodeRegDnaseClustered/). The remaining regions were divided into Ikb fragments. 90 such Ikb fragments were sampled from each chromosome. Fragments containing >=20 consecutive N's were removed. The remaining sequences were submitted to CRISPick run under CRISPRi settings. The CRISPick output was further filtered for spacers that meet these criteria: 1) off-target prediction criteria described in the beginning of this section, and 2) On-target Efficacy Score >= 0.5 (the rationale is to maximize representation by likely active crRNAs to bias for revealing any potential cell fitness effects from non-specific genotoxicity due to residual DNA cutting by multiCasl2a- KRAB), 3) mapping uniquely to the hgl9 genome by Bowtie (Langmead et al. 2009) using '-m 1' and otherwise default parameters, 3) filtered once more against those whose uniquely mapped site falls within lOkb of annotated ensembl genes or any ENCODE DNase hypersensitive site. [00166] Non-targeting negative control spacers were generated by combining 1) nontargeting negative controls in the Humagne C and D libraries, 2) taking 20nt non-targeting spacers from the dCas9-KRAB CRISPRi_v2 genome-wide library (Horlbeck et al. 2016a), removing the G in the 1st position, and appending random 4-mers to the 3' end. This set of spacers were then filtered for those that do not map to the hgl9 genome using Bowtie with default settings. Library 2 (6-plex cRNA’s)

[00167] Sublibrary A (84,275 constructs): Test position spacers were encoded at each position of the 6-plex array, with remaining positions referred to as context positions and filled with negative control spacers. Test positions can encodes one of 506 intergenic negative control spacers and 2,303 essential TSS-targeting spacers. The essential TSS-targeting spacers were selected from among all spacers targeting PAM's within -50bp to +300bp TSS-proximal regions of 50 common essential genes with the strongest K562 cell fitness defect in prior dCas9-KRAB CRISPRi screen (Horlbeck et al. 2016a), and must have >0.7 CRISPick On-target Efficacy Score. Negative control context spacers consist of 5 6-plex combinations, 3 of these combinations consist entirely of non-targeting negative controls and 2 of the combinations consist entirely of intergenic negative controls.

[00168] Sublibrary B (6,370 constructs): crRNA combinations targeting cis-regulatory elements at the MYC locus were assembled from a subset of combinations possible from 15 starting spacers (3 targeting MYC TSS, 3 targeting each of 3 enhancers, and 3 intergenic negative control spacers). The 3 enhancer elements are described in the subsection "Design of individual crRNAs." These 15 starting spacers were grouped into 5 3-plex combinations, each 3-plex combination exclusively targeting one of the 4 cis-regulatory elements, or consisting entirely of intergenic negative controls. Each 3-plex was then encoded in positions 1-3 of 6-plex arrays, and positions 4-6 were filled with all possible 3-plex combinations chosen from the starting 15 spacers. All 6-plex combinations were also encoded in the reverse order in the array. [00169] All-negative control constructs (2000 constructs): 1500 6-plex combinations were randomly sampled from the intergenic negative control spacers described for Library 1. 5006-plex combinations were randomly sampled from non-targeting negative control spacers described for Library 1.

[00170] Intergenic negative controls and non-targeting negative controls are defined the same as in Library 1.

[00171 ] crRNA library construction. For Library 1, -140 fmol of pooled oligo libraries from Twist were subjected to 10 cycles of PCR amplification using primers specific to adaptor sequences flanking the oligos and containing BsmbI sites. The PCR amplicons were cloned into a crRNA expression backbone (pCH67) by Golden Gate Assembly with -1: 1 insertbackbone ratio using -500 fmol each. Golden Gate Assembly reaction was earned out in a lOOul reaction containing 2.5U Esp3I (Thermo ER0452) and 1000U T4 DNA Ligase in T4 DNA Ligase reaction buffer (NEB M0202L). The reaction mix was incubated for 31 cycles alternating between 37deg. C and 16deg. C for 20min at each temperature, then heat-inactivated at 65deg. C for 5min. Assembly reactions were column purified with Zymo DNA clean and concentrator- 5 (Zymo D4004), eluted in 12ul of water and <7ul added to 70ul of MegaX DH10B T1R Electrocomp Cells (C640003) for electroporation using BioRad Gene Pulser Xcell Electroporator with settings 2.0kV, 200ohms, 25pF. Cells were recovered at 37deg. C for rotating for Ih in ~5ml recovery media from the MegaX DH10B T1R Electrocomp Cells kit and small volumes plated onto bacterial LB plates containing carbenicillin for quantification of colony forming units. The remaining recovery culture was inoculated directly into 200ml liquid LB media with carbenicillin and incubated in 37deg. C shaker for 12h-16h prior to harvesting for plasmid purification using ZymoPURE II Plasmid Midiprep kit (Zymo D4200). Based on the colony forming units from the small volumes in the bacterial plates, the estimated coverage of the library is 778x. 24 individual colonics were verified by Sanger sequencing and the library subjected to deep sequencing as described in Illumina sequencing library preparation. For Library 2, 915 fmol of pooled oligo libraries from Twist was subjected to 18 cycles of PCR amplification and agarose gel purification of the correctly sized band before proceeding similarly with the remainder of the protocol as described above. The estimated coverage of the library from colony forming units is ~60x.

[00172] Illumina sequencing library preparation. crRNA inserts were amplified from genomic DNA isolated from screens using 16 cycles of first round PCR using pooled 0-8nt staggered forward and reverse primers, treated with ExoSAP-IT (Thermo Fisher 78201. l.ML), followed by second round of PCR to introduce Illumina unique dual indices and adaptors. Sequencing primer binding sites, unique dual indices, P5 and P7 adaptor sequences are from Illumina Adaptor Sequences Document # 1000000002694 vl6. PCR amplicons were subject to size selection by magnetic beads (SPRIselect, Beckman B23318) prior to sequencing on an Illumina NovaSeq6000 using SP100 kit for Library 1 or SP500 kit for Library 2. Sequencing of plasmid libraries were performed similarly, except 7 cycles of amplification were each used for Round 1 and Round 2 PCR. The size distribution of the final library was measured on an Agilent TapeStation system. We noted that even after magnetic bead selection of Round 2 PCR- amplified Library 2 plasmid library (colonies from which were Sanger sequencing verified) and genomic DNA from screens, smaller sized fragments from non-specific PCR amplification during Illumina sequencing library preparation persisted. This might contribute to the fraction of reads that could not be mapped to our reference 6-plex array. Thus, these unmapped reads do not necessarily reflect recombination of the crRNA library constructs, though the latter could contribute as well.

[00173] Cell fitness screens. Library 1 screen: K562 cells engineered by piggyBac transposition to constitutively express denAsCasl2a-KRAB or multiAsCasl2a-KRAB were transduced with lentivirally packaged Library 1 constructs at MOI = -0.15. Transduced cells were then selected using lug/ml puromycin for 2 days, followed by washout of puromycin. On Day 6 after transduction, initial (TO) time point was harvested, and the culture was split into 2 replicates that are separately cultured henceforth. 10 days later (T10), the final time point was harvested (8.6 total doublings for multiAsCasl2a-KRAB cells, 9.15 total doublings for denasCasl2a-KRAB cells). A cell coverage of >500x was maintained throughout the screen. Library 2 screen: K562 cells engineered by piggyBac transposition to constitutively express multiAsCasl2a-KRAB were transduduced with lentivirally packaged Library 2 constructs at MOI = -0.15. The screen was carried out similarly as described for Library 1 screen, except the screen was carried out for 14 days (T14) or 13.5 total doublings and maintained at a cell coverage of >2000x throughout. Genomic DNA was isolated using the NucleoSpin Blood XL Maxi kit (Machery-Nagel 740950.50).

[00174] Screen data processing and analysis. Summary of library contents are in FIG. 24. Library 1: Reads were mapped to crRNA constructs using sgcount (https://noamteyssier.github.io/sgcount/), requiring perfect match to the reference sequence. [00175] Library 2: First, reference construct sequences were created by interspersing provided spacer and constant regions. Each construct is then given a unique construct id (CID). Each CID is then split into R1 and R2 reference sequences, which are constructed by taking the first three and last three spacer-construct pairs of the reference sequence respectively. The R2 sequence is then reverse complemented for matching against the R2 sequencing reads. Next, two hashmaps are created for the R1 and R2 spacer-construct pairs respectively, which map the R1/R2 sequences to a set of corresponding CIDs. Finally, for each R1/R2 sequencing pair, each kmer (k = length of R1/R2 respective construct sequence) in the sequence is mapped against their respective R1/R2 hashmap. If both sequencing pairs are able to be mapped to a CID set. then the intersection of their sets is their original construct, and the total count of that CID is incremented. The algorithm is implemented in Rust and is available at https://github.com/noamteyssier/casmap .

[00176] Starling from read counts, the remainder of analyses were performed using custom scripts in R. Constructs that contained <1 read per million reads (RPM) aligned to the reference library in either replicates at TO were removed from analysis. From the constructs that meet this read coverage threshold, a pseudocount of 1 was added for each construct and the RPM re-calculated and used to obtain a fitness score (Kampmann et al. 2013): where RPM = read count per million reads mapped to reference (initial = at TO, final = at end of screen), negctrlmedian = median of RPM of intergenic negative control constructs, totaldoublings = total cell population doublings in the screen. For Library 1, data from a single TO sample was used to calculate the fitness score for both replicates due to an unexpected global loss of sequencing read counts for one of two originally intended TO replicate samples.

[00177] 3' RNA-seq experiment and data analysis

[00178] Experimental procedure. 3' RNA-seq was performed as part of a batch processed using a QuantSeq-Pool Sample-Barcoded 3' mRNA-Seq Library Prep Kit for Illumina (Lexogen cat#139) in accordance with the manufacturer’s instructions. Briefly, 10 ng of each purified input RNA was used for first strand cDNA synthesis with an oligo(dT) primer containing a sample barcode and a unique molecular identifier. Subsequently, barcoded samples were pooled and used for second strand synthesis and library amplification. Amplified libraries were sequenced on an Illumina HiSeq4000 with 100 bp paired-end reads. The QuantSeq Pool data was demultiplexed and preprocessed using an implementation of pipeline originally provided by Lexogen (https://github.com/Lexogen-Tools/quantseqpool_analysis). The final outputs of this step are gene level counts for all samples (including samples from multiple projects multiplexed together).

[00179] Gene level and differential expression analysis. For generating scatter plots, nomiTransform function from DESeq2 (Love et al. 2014) used to normalize the raw counts and then a pseudocount of 1 added, and log2-transformed. The output was plotted in R as scatter plots. [00180] For differential expression analysis, DESeq2 (version 1.34) default Wald-test was used to compare each targeting construct (one replicate) with non-targeting samples (two replicates). We calculated log2 transformed TPM counts and applied the threshold of 6.5 to eliminate genes with low expression. Using ggplot2, volcano plots visualized in R are then displayed for genes with log2FoldChange above or below 2.055 and p-values smaller than 0.01. The log2FoldChange cutoff was based on visually examining the concordance between two replicates of untransduced controls and manually identifying a threshold below which the log2FoldChange are poorly correlated between the replicates of the untransduced control.

[00181 ] Off-largel analysis of spacers. To evaluate potential off-target effect of spacers, we used the crisprVerse (version 1.0.0) (Hoberecht et al. 2022) and crisprBowtie (version 1.2.0) together with other R packages including GenomicRanges (version 1.50) (Lawrence et al. 2013) and tidyverse (version 1.3.2) (Wickham et al. 2019). First, we defined dictionary of spacers as "TCCTCCAGCATCTTCCACATTCA":"HBG-2", (SEQ ID NO: 5) "TTCTTCATCCCTAGCCAGCCGCC":"HBG-3", (SEQ ID NO: 6) "CTTAGAAGGTTACACAGAACCAG":"HS2-1", (SEQ ID NO: 7) "TGTGTAACCTTCTAAGCAAACCT":"HS2-2", (SEQ ID NO: 8) "AGGTGGAGTTTTAGTCAGGTGGT":"HS2-3", (SEQ ID NO: 9) "ATTAACTGATGCGTAAGGAGATC":"NT-3" (SEQ ID NO: 10). Then, 'runCrisprBowtie' function used with these parameters: 'crisprNuclease' as 'enAsCasl2a', 'n_mismatches' equal 3. 'canonical' equal FALSE, and 'bowtie_index' as a path to folder including pre-indexed hg38 reference genome. Thus, the results from this step allows us to assess our previously designed spacers and annotate potential off-target loci in the human genome. To annotate results, we used reference annotation GENCODE (version 34) (Frankish et al. 2021) and we defined pam_site +/- 2500 bp for each predicted off-target to overlap them with matched transcription start sites (TSS) +/- 1000 bp of all annotated genes. Results shown as annotated tables.

[00182] RT-qPCR. For the CRISPRi experiments targeting the HBG TSS or HS2 enhancer, K562 cells engineered (by lentiviral transduction at MOI ~5) for constitutive expression of multiAsCasl2a-KRAB were transduced with crRNAs and sorted, followed by resuspension of ~200k to 1 million cells in 300ul RNA Lysis Buffer from the Quick-RNA Miniprep Kit (Zymo R1O55) and stored in -70deg. C. RNA isolation was performed following the kit's protocols, including on-column DNase I digestion. 500ng of RNA was used as input for cDNA synthesis primed by random hexamers using the RevertAid RT Reverse Transcription Kit (Thermo fisher K1691), as per manufacturer's instructions. cDNA was diluted 1:4 with water and 2ul used as template for qPCR using 250nM primers using the SsoFast EvaGreen Supermix (BioRad 1725200) on an Applied Biosystems ViiA 7 Real Time PCR System. Data was analyzed using the ddCT method, normalized to GAPDH and no crRNA sample as reference.

[00183] Transient transfection experiments. For co-transfection experiments, transfections were performed similar to prior study (Campa et al. 2019). Briefly, the day before transfection, 100,000 HEK293T cells were seeded into wells of a 24 well plate. The following day, we transiently transfected 0.6pg of each protein construct and 0.3pg gRNA construct per well (in duplicate) in Mirus TransIT-LTl transfection reagent according to manufacturer’s instructions. Mixtures were incubated at room temperature for 30 minutes and then added in dropwise fashion into each well. 24 hours after transfection, cells were replenished with fresh media. 48 hours after transfection, BFP and GFP positive cells (indicative of successful delivery of protein and crRNA constructs) were sorted (BD FACSAria Fusion) and canned out for subsequent flow-cytometry experiments.

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[00184] Notwithstanding the appended claims, the disclosure set forth herein is also described by the following clauses: 1. A method for transcriptionally silencing genomic target sites in a cell, the method comprising: contacting a cell with: a first expression cassette comprising a nucleic acid encoding a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptide operably linked to a promoter, wherein the variant type V CRISPR/Cas effector polypeptide comprises a R1226A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide and the one or more heterologous polypeptide is a transcriptional repression domain; and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA), thereby transcriptionally silencing genomic target sites in the cell.

2. The method of clause 1, wherein the first expression cassette is contained in a lentivirus expression vector.

3. The method of clauses 1 or 2, wherein the second expression cassette is contained in a lentivirus expression vector.

4. The method of clauses 2 or 3, wherein the first and the second expression cassettes are contained in the same lentivirus expression vector.

5. The method of any of clauses 1-4, wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide. 6. The method of any of clauses 1-5, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

7. The method of clauses 5 or 6, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

8. The method of clauses 5-7, wherein the variant type V CRISPR/Cas effector polypeptide comprises a E174R a amino acid substitution, a S542R amino acid substitution, and a K548R amino acid substitution, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

9. The method of any of clauses 1-8, wherein the promoter of the second expression cassette is an EF- la promoter.

10. The method of any of clauses 1-9, wherein the antibiotic resistance gene of the second expression cassette is an antibiotic resistance gene that provides resistance to puromycin.

11. The method of any of clauses 1-10, wherein the 3’ LTR of the second expression cassette comprises 2 to 10 gRNAs.

12. The method of any of clauses 1-11, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 400bp or more.

13. The method of any of clauses 1-12, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 700bp or more.

14. The method of any of clauses 1-13, wherein the contacting results in transcriptional silencing of 1 or more target genomic sites. 15. The method of any of clauses 1-14, wherein the contacting results in transcriptional silencing of 5 or more target genomic sites when the first and second expression cassettes are expressed as a single copy in the cell.

16. The method of any of clauses 1-15, wherein the transcriptional repressor domain is a Kruppel-associated box (KRAB) domain.

17. The method of any of clauses 1-16, wherein the one or more guide RNAs hybridize near transcriptional start sites (TSS).

18. A method for transcriptionally activating genomic target sites in a cell, the method comprising: contacting a cell with: a first expression cassette comprising a nucleic acid encoding a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptide operably linked to a promoter, wherein the variant type V CRISPR/Cas effector polypeptide comprises a R1226A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide and the one or more heterologous polypeptide is a transcriptional activation domain; and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA), thereby transcriptionally activating genomic target sites in the cell. 19. The method of clause 18, wherein the first expression cassette is contained in a lentivirus expression vector.

20. The method of clauses 18 or 19, wherein the second expression cassette is contained in a lentivirus expression vector.

21. The method of clauses 19 or 20. wherein the first and the second expression cassettes are contained in the same lentivirus expression vector.

22. The method of any of clauses 18-21, wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide.

23. The method of any of clauses 18-22, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

24. The method of clauses 22 or 23, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

25. The method of clauses 22-23, wherein the variant type V CRISPR/Cas effector polypeptide comprises a E174R amino acid substitution, a S542R amino acid substitution, and a K548R amino acid substitution, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

26. The method of any of clauses 22-25, wherein the promoter of the second expression cassette is an EF-la promoter.

27. The method of any of clauses 18-26, wherein the antibiotic resistance gene of the second expression cassette is an antibiotic resistance gene that provides resistance to puromycin. 28. The method of any of clauses 18-27, wherein the 3’ LTR of the second expression cassette comprises 2 to 10 gRNAs.

29. The method of any of clauses 18-28, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 400bp or more.

30. The method of any of clauses 18-29, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 700bp or more.

31. The method of any of clauses 18-30, wherein the contacting results in transcriptional activation of 1 or more target genomic sites.

32. The method of any of clauses 18-31, wherein the contacting results in transcriptional activation of 2 or more target genomic sites when the first and second expression cassettes are expressed as a single copy in the cell.

33. The method of any of clauses 18-32, wherein the transcriptional activation domain is selected from the group consisting of a VP64 domain, a p65 domain, a Rta domain, an AD2 domain, a CR3 domain, an EKLF1 domain, a GATA4 domain, a PR VIE domain, a p53 domain, a SP1, a MYOD, MEF2C, a TAX domain, a PPARg domain, a MED1 domain, a MED7 domain, a MED26 domain, a MED29 domain, a TBP domain, a GTF2H-sD domain and a GTF2B domain.

34. The method of any of clauses 18-33, wherein the one or more guide RNAs hybridize near transcriptional start sites (TSS).

35. A method for epigenetically modifying genomic target sites in a cell, the method comprising: contacting a cell with: a first expression cassette comprising a nucleic acid encoding a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides operably linked to a promoter, wherein the variant type V CRISPR/Cas effector polypeptide comprises a R1226A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide and the one or more heterologous polypeptide is an epigenetic modification domain; and a second expression cassette comprising a nucleic comprising the following in order: a 5’ long terminal repeat (LTR), a promoter, an antibiotic resistance gene, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a 3’ LTR wherein the 3’ LTR comprises a U6 promoter and one or more guide RNAs (gRNA), thereby epigenetically modifying genomic target sites in the cell.

36. The method of clause 35, wherein the first expression cassette is contained in a lentivirus expression vector.

37. The method of clauses 35 or 36, wherein the second expression cassette is contained in a lentivirus expression vector.

38. The method of clauses 36 or 37, wherein the first and the second expression cassettes are contained in the same lentivirus expression vector.

39. The method of any of clauses 35-38, wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide. 40. The method of any of clauses 35-39, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

41. The method of clauses 39 or 40, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

42. The method of clauses 39-41, wherein the variant type V CRISPR/Cas effector polypeptide comprises a E174R amino acid substitution, a S542R amino acid substitution, and a K548R amino acid substitution, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

43. The method of any of clauses 35-42, wherein the promoter of the second expression cassette is an EF-la promoter.

44. The method of any of clauses 35-43, wherein the antibiotic resistance gene of the second expression cassette is an antibiotic resistance gene that provides resistance to puromycin.

45. The method of any of clauses 35-44, wherein the 3’ LTR of the second expression cassette comprises 2 to 10 gRNAs.

46. The method of any of clauses 35-45, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 400bp or more.

47. The method of any of clauses 35-46, wherein the length of the U6 promoter and the 1 or more gRNAs in the 3’ LTR of the second expression cassette is 700bp or more.

48. The method of any of clauses 35-47, wherein the contacting results in epigenetic modification of 1 or more target genomic sites. 49. The method of any of clauses 35-48, wherein the contacting results in epigenetic modification of 1 or more target genomic sites when the first and second expression cassettes are expressed as a single copy in the cell.

50. The method of any of clauses 35-49, wherein the epigenetic modification domain is selected from the group consisting of a DNA methyltransferase, a DNA demethylase domain, a histone methyltransferase domain, and a histone demethylase domain.

51. A nucleic acid comprising the second expression cassette of any of the preceding clauses.

52. A nucleic acid comprising the first and the second expression cassette of any of the preceding clauses.

53. A recombinant expression vector comprising a nucleic acid comprising the second expression cassette of any of the preceding clauses.

54. A recombinant expression vector comprising a nucleic acid comprising the first and the second expression cassette of any of the preceding clauses.

55. The recombinant expression vector of clauses 53 or 54, wherein the recombinant expression vector is a viral vector.

56. The recombinant expression vector of clause 55, wherein the viral vector is a lentivirus expression vector.

57. A system comprising a variant type V CRISPR/Cas effector polypeptide and the recombinant expression vector of clause 53, wherein the variant type V CRISPR/Cas effector polypeptide comprises a R 1226 A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide. 58. The system of clause 57, wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide.

59. The system of clauses 57 or 58, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

60. The system of any of clauses 57-59, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

60. The system of any of clauses 57-60, wherein the variant type V CRISPR/Cas effector polypeptide is fused to one or more heterologous polypeptide.

61. The system of any of clauses 57-60, wherein the recombinant expression vector is a lentiviral vector.

62. A method for transcriptionally modulating genomic target sites in a cell, the method comprising: contacting a cell with: a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides, wherein the variant type V CRISPR/Cas effector polypeptide comprises a nickase mutation and the one or more heterologous polypeptide comprise a transcriptional modulation domain; and one or more guide RNAs (gRNA), thereby transcriptionally modulating genomic target sites in the cell.

63. The method of clause 62 wherein the valiant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide. 64. The method of clauses 62 or 63, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

65. The method of any of clauses 62-64, wherein the nickase mutation is a substitution of a arginine at position 1226 of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide with an alanine

66. The method of any of clauses 62-65, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

67. The method of any of clauses 62-66, wherein the variant type V CRISPR/Cas effector polypeptide comprises a E174R amino acid substitution, a S542R amino acid substitution, and a K548R amino acid substitution, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

68. The method of any of clauses 62-67, wherein the transcriptional modulation is transcriptional silencing.

69. The method of clause 68, wherein the one or more heterologous polypeptides comprises a transcriptional repression domain.

70. The method of clause 69, wherein the transcriptional repression domain is selected from the group consisting of DMNT1, SET1, HDAC11, DMNT3A, SETD8, EZH2, SUV39H1, PHF19, SALE NUE, SUVR4, KYP. DIM5. HDAC8, SIRT3, SIRT6. MESOLO4. SET8, HST2, COBB, SET-TAF1B, NCOR, MeCP2 SIN3A, HDT1, MBD2B, NIPP1, HP1A, KRAB, and any combination thereof.

71. The method of any of clauses 62-67, wherein the transcriptional modulation is transcriptional activation. 72. The method of clause 71, wherein the one or more heterologous polypeptides comprises a transcriptional activation domain.

73. The method of clause 72, wherein the transcriptional activation domain is selected from the group consisting of DMNT1, SET1, HDAC11, DMNT3A, SETD8, EZH2, SUV39H1, PHF19, SALE NUE, SUVR4, KYP. DIM5. HDAC8, SIRT3, SIRT6. MES0L04. SET8, HST2, COBB, SET-TAF1B, NCOR, MeCP2 SIN3A, HDT1, MBD2B, NIPP1, HP1A, KRAB, and any combination thereof.

74. The method of any of clauses 62-73, wherein the one or more gRNAs are two to ten gRNAs.

75. The method of any of clauses 62-74, wherein the variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides is encoded by a nucleic acid.

76. The method of clause 75, wherein the nucleic acid is RNA.

77. The method of clause 75, wherein the nucleic acid is a first expression cassette comprising a nucleic acid encoding a variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide operably linked to a promoter.

78. The method of any of clauses 62-75, wherein the one or more gRNAs are encoded by a second expression cassette comprising a nucleic acid comprising the one or more gRNAs operable linked to a promoter.

79. The method of clause 78, wherein the second expression cassette further comprises a 5’ long terminal repeat (LTR) and a 3’LTR, wherein the 3’ LTR comprises the one or more gRNAs operable linked to the promoter. 80. The method of any of clauses 62-79, wherein the contacting results in transcriptional modulation of 5 or more target genomic sites.

81. The method of any of clauses 62-80, wherein the one or more guide RNAs hybridize near transcriptional start sites (TSS) of coding genes or non-coding genomic elements.

82. A method for epigenetically modifying genomic target sites in a cell, the method comprising: contacting a cell with: a variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides, wherein the variant type V CRISPR/Cas effector polypeptide comprises a nickase mutation and the heterologous polypeptide is an epigenetic modification domain; and one or more guide RNAs (gRNA), epigenetically modifying genomic target sites in the cell.

83. The method of clause 82 wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide.

84. The method of clauses 82 or 83, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

85. The method of any of clauses 82-84, wherein the nickase mutation is a substitution of an arginine at position 1226 of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide with an alanine

86. The method of any of clauses 82-85, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide. 87. The method of any of clauses 82-86, wherein the variant type V CRISPR/Cas effector polypeptide comprises a E174R amino acid substitution, a S542R amino acid substitution, and a K548R amino acid substitution, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

88. The method of any of clauses 82-87, wherein the one or more gRNAs are two to ten gRNAs.

89. The method of any of clauses 82-88, wherein the variant type V CRISPR/Cas effector polypeptide fused to one or more heterologous polypeptides is encoded by a nucleic acid.

90. The method of clause 89, wherein the nucleic acid is RNA.

91. The method of clause 89, wherein the nucleic acid is a first expression cassette comprising a nucleic acid encoding a variant type V CRISPR/Cas effector polypeptide fused to a heterologous polypeptide operably linked to a promoter.

92. The method of any of clauses 82-89, wherein the one or more gRNAs are encoded by a second expression cassette comprising a nucleic acid comprising the one or more gRNAs operable linked to a promoter.

93. The method of clause 92, wherein the second expression cassette further comprises a 5’ long terminal repeat (LTR) and a 3’LTR, wherein the 3’ LTR comprises the one or more gRNAs operable linked to the promoter.

94. The method of any of clauses 82-93, wherein the contacting results in epigenetic modification of 1 or more target genomic sites.

95. The method of any of clauses 82-94, wherein the contacting results in epigenetic modification of 5 or more target genomic sites. 96. The method of any of clauses 82-94, wherein the one or more guide RNAs hybridize near transcriptional start sites (TSS) of coding genes or non-coding genomic elements.

97. The method of any of clauses 82-96, wherein the epigenetic modification domain is selected from the group consisting of a DNA methyltransferase, a DNA demethylase domain, a histone methyltransferase domain, and a histone demethylase domain.

98. A nucleic acid comprising the second expression cassette of any of clauses 78-81 or 92-97.

99. A nucleic acid comprising the first and the second expression cassette of any of clauses 77-81 or 91-97.

100. A recombinant expression vector comprising a nucleic acid comprising the second expression cassette of any of clauses 78-81 or 92-97.

101. A recombinant expression vector comprising a nucleic acid comprising the first and the second expression cassette of any of clauses 77-81 or 91-97.

102. The recombinant expression vector of clauses 100 or 101, wherein the recombinant expression vector is a viral vector.

103. The recombinant expression vector of clause 102, wherein the viral vector is a lentivirus expression vector.

104. A system comprising a variant type V CRISPR/Cas effector polypeptide and the recombinant expression vector of clause 100, wherein the variant type V CRISPR/Cas effector polypeptide comprises a R 1226 A amino acid substitution of SEQ ID NO: 1 or a corresponding position in another type V CRISPR/Cas effector polypeptide. 105. The system of clause 104, wherein the variant type V CRISPR/Cas effector polypeptide is an Acidaminococcus sp. variant type V CRISPR/Cas effector polypeptide.

106. The system of clauses 104 or 105, wherein the variant type V CRISPR/Cas effector polypeptide is a Casl2a CRISPR/Cas effector polypeptide.

107. The system of any of clauses 104-106, wherein the variant type V CRISPR/Cas effector polypeptide further comprises a E174R amino acid substitution, a S542R amino acid substitution, a K548R amino acid substitution, a combination thereof, or a corresponding position in another type V CRISPR/Cas effector polypeptide.

108. The system of any of clauses 104-107, wherein the variant type V CRISPR/Cas effector polypeptide is fused to one or more heterologous polypeptides.

109. The system of any of clauses 104-108, wherein the recombinant expression vector is a lentiviral vector.