Tailoring a CRISPR/Cas-based Epigenome Editor for Programmable Chromatin Acylation and Decreased Cytotoxicity

Abstract

Engineering histone acylation states can inform mechanistic epigenetics and catalyze therapeutic epigenome editing opportunities. Here, we developed engineered lysine acyltransferases that enable the programmable deposition of acetylation and longer-chain acylations. We show that targeting an engineered lysine crotonyltransferase results in weak levels of endogenous enhancer activation yet retains potency when targeted to promoters. We further identify a single mutation within the catalytic core of human p300 that preserves enzymatic activity while substantially reducing cytotoxicity, enabling improved viral delivery. We leveraged these capabilities to perform single-cell CRISPR activation screening and map enhancers to the genes they regulate in situ. We also discover acylation-specific interactions and find that recruitment of p300, regardless of catalytic activity, to prime editing sites can improve editing efficiency. These new programmable epigenome editing tools and insights expand our ability to understand the mechanistic role of lysine acylation in epigenetic and cellular processes and perform functional genomic screens.

Keywords: CRISPR; CRISPRa; EP300; Epigenome editing; Perturb-seq; enhancer; histone acetylation; histone crotonylation; off-target editing; protein engineering.

Figure 1:. Targeting of p300 variants to OCT4 regulatory elements results in divergent transcriptional responses.

(A) Construct designs for testing dCas9-p300 variants. (B) Western blot analysis of whole proteome lysine crotonylation probed using anti-KCr antibody in HEK293T cells overexpressing dCas9-p300 variant. (C) In vitro acylation activity assay of dCas9-p300 variants measured using the acyl-CoA co-factor indicated on the X and Y axes. Activity was measured using the N-terminal H3 peptide (n = 5, mean ± sem). (D) Top: Schematic of the OCT4 locus with red rectangles indicating target gRNA location relative to transcription start site. Bottom: RT-qPCR of OCT4 gene expression upon dCas9-p300 variant targeting in HEK293T cells (n = 3, mean ± sem). (E) Relative enrichment of indicated histone modification measured by CUT&RUN-qPCR following delivery of dCas9-p300 variant and OCT4 distal enhancer targeting gRNAs in HEK293T cells (n = 4–6, mean ± sem). (F) Same as (E) with OCT4 promoter targeting gRNAs (n = 4–6, mean ± sem).

Figure 2:. Developing a high throughput screen to assess protein expression and transcriptional activity in mammalian cells.

(A) Schematic of the screening pipeline. A reporter gene is stably integrated into the AAVS1 locus with 5x upstream TetO sites in HEK293T cells. A library of p300 variants fused to rTetR is transduced into the reporter cell line, selected with blasticidin, and induced with doxycycline for 48 hours before sorting and sequencing. (B) PDB (5LKU) crystal structure of the p300 core domain bound to Coenzyme A. (C) Citrine and mCherry fluorescence levels of an individual denoted variant of rTetr-p300 were measured by flow cytometry 11 days after transduction. (D) Heatmap illustrating effects of all single mutations on citrine activation reporter. Deleterious mutations are denoted in magenta and beneficial mutations in green. Squares in yellow did not appear in the screening results. Ratios are shifted to the median of negative degenerate sequence controls (n=2). (E) Heatmap illustrating effects of all single mutations on mCherry expression reporter. Deleterious mutations are denoted in black and beneficial mutations in red. Squares in yellow did not appear in the screening results. Ratios are shifted to the median of negative degenerate sequence controls (n=2). (F) Top: Construct design for testing epigenome effector domains protein stability against mRNA expression levels. Bottom: Representative flow cytometry plots of mRNA expression (mChe) across 4 different epigenome editor effector domains with and without catalytic activity.

Figure 3:. I1417N mutation reduces p300 cytotoxicity and improves viral delivery while preserving activity

(A) Scatter plot depicting the relationship between reporter activation (x-axis) and protein expression (y-axis). In green/magenta are the top 100/bottom variants summing the log ratios of both fluorescent readouts. Hits from shaded regions were selected for individual validation. (B) % of mCherry-positive cells across each single construct validation in transient transfection measured by flow cytometry. (C) RT-qPCR of HBG1 gene expression upon dCas9-p300 variant targeting to HBG1 (x-axis) and HS2 enhancer (y-axis) (n = 3, mean ± sem). (D) RT-qPCR of OCT4 gene expression upon dCas9-p300 variant targeting across OCT4 distal enhancer, proximal enhancer, and promoter, respectively (n = 3, mean ± sem). (E) Western blot analysis of HEK293T cells overexpressing dCas9-p300 variant probing for anti-Cas9 with anti-Tubulin as loading control. (F) Fluorescence microscopy image of HEK293T cells transiently transfected with dCas9-p300 variant bicistronically expressed with mCherry and scrambled gRNA. Top panel: mCherry channel fluorescence. Bottom panels: DAPI staining. Scale bar: 100um. (G) Cell viability as measured using alamarBlue^™ cell viability reagent 48 hours post-transient transfection with dCas9-p300 variant and scrambled gRNA in HEK293T cells. (H) % of mCherry-positive cells in 2%v/v transduced K562 cells measured by flow cytometry. (I) RT-qPCR of HBG1 gene expression upon dCas9-p300 or CBP variant targeting to HBG1 (x-axis) and HS2 enhancer (y-axis) (n = 3, mean ± sem).

Figure 4:. Immunoprecipitation-mass spectrometry (IP-MS) reveals different classes of protein interactions within different p300 variants.

(A) Venn diagram of hits called in IP-MS experiment (n = 2, P < 0.05) (B) Dot chart showing top 84 protein hits by greatest -log(P-value) relative to dCas9 only bait. Classes of interactions are hierarchically clustered and subgroups are named on characteristic similarity. (C) Volcano plot of the protein interactome of the p300 I1417N variant relative to the wild type. Red highlights correspond to P < 0.05 (n = 2) (D) Gene ontology analysis of 1417N-enriched hits. (E) Percentage editing as measured by BFP+ cells in GFP-BFP conversion assay or % editing via prime editing co-transfected with epigenome editor targeting the same site in HEK293T cells (n = 2–3, mean ± sem).

Figure 5.. dCas9-p300 I1417N has reduced off-targeting activity.

(A) Transcriptomes of HEK293T cells transiently expressing dCas9-p300 WT compared against dCas9 (n = 2 biological replicates). Each black dot indicates the expression level of an individual gene. Red dashed lines indicate the 2x difference between sample groups. (B) Genomic coordinates spanning ~5,248,000 to ~ 5,255,256 bp of human chromosome 11 (GRCh38/hg38) are shown along with H2BK20ac, H3K27ac, H3K4me3, and H3K27me3 CUT&RUN enrichment data with the indicated dCas9-p300 variant and 4 HBG1-targeting sgRNAs in HEK293T cells (n = 2 biological replicates). (C) Profile of H3K27ac enrichment at transcription start sites. (D-F) Relative enrichment of indicated histone modification (H2BK20ac, H3K4me3, or H3K27me3) measured by CUT&RUN-qPCR following delivery of dCas9-p300 variant and HBG1 promoter targeting gRNAs in HEK293T cells (n = 4, mean ± sem).

Figure 6.. A high MOI Perturb-seq screen to identify effector-specific cis regulatory elements.

(A) Schematic of the experimental framework. Monoclonal KS62 cells expressing dCas9-p300 fusions were transduced at high MOI (~20) to introduce sgRNAs targeting regulatory elements of interest followed by scRNA-seq. (B) Violin plots of expression change over the null control of all statistically significant hits across p300 WT and p300 I1417N (n=48 and 24 hits, respectively). (C-D) Volcano plot shows the statistically significant hits of each effector. Red highlights correspond to P < 0.01. An FDR of 0.10 was used after a Benjamini-Hochberg correction was used to call hits. (E-F) Violin plots of normalized expression levels compared to the null control of select genes across p300 WT and p300 I1417N targeting promoters/enhancers.