Programmable epigenome editing by transient delivery of CRISPR epigenome editor ribonucleoproteins

Abstract

Programmable epigenome editors modify gene expression in mammalian cells by altering the local chromatin environment at target loci without inducing DNA breaks. However, the large size of CRISPR-based epigenome editors poses a challenge to their broad use in biomedical research and as future therapies. Here, we present Robust ENveloped Delivery of Epigenome-editor Ribonucleoproteins (RENDER) for transiently delivering programmable epigenetic repressors (CRISPRi, DNMT3A-3L-dCas9, CRISPRoff) and activator (TET1-dCas9) as ribonucleoprotein complexes into human cells to modulate gene expression. After rational engineering, we show that RENDER induces durable epigenetic silencing of endogenous genes across various human cell types, including primary T cells. Additionally, we apply RENDER to epigenetically repress endogenous genes in human stem cell-derived neurons, including the reduction of the neurodegenerative disease associated V337M-mutated Tau protein. Together, our RENDER platform advances the delivery of CRISPR-based epigenome editors into human cells, broadening the use of epigenome editing in fundamental research and therapeutic applications.

Fig. 1. Virus-like particle delivery of epigenome editor RNPs.

a Schematic of a virus-like particle packaging epigenome editors. b Schematic overview of different CRISPR-based epigenome editors and their sizes. kb; kilobase pairs. cS. pyogenes Cas9 (spCas9) protein quantifications of CRISPRoff-eVLP collected at 48 h and 72 h post-transfection. Protein contents were measured by anti-Cas9 ELISA. Data are shown as individual data points and mean ± SD for n = 3 biological replicates. d Western blot evaluating the packaging of CRISPRoff protein in eVLPs at 4, 2 and 1 μl of concentrated CRISPRoff-eVLP with anti-Flag antibody. CRoff; CRISPRoff. e Schematic of engineered virus-like particle transduction and CLTA-GFP silencing in HEK293T reporter cells. f Quantification of CLTA-GFP silencing in HEK293T cells 3 days post-treatment with different epigenome editor eVLPs (16 μl). g Representative flow cytometry plots of CLTA-GFP expression in HEK293T cells at day 3 and 14 post-treatment with CRISPRi-eVLPs and CRISPRoff-eVLPs (16 μl). h Time course of CLTA-GFP silencing in HEK293T cells post-treatment with different epigenome editor eVLPs (16 μl). Values and error bars reflect mean ± SD of n = 3 biological replicates. i Schematic of engineered virus-like particle transduction and CLTA-GFP reactivation in HEK293T reporter cells. j Time course of CLTA-GFP reactivation in HEK293T cells post-treatment with RENDER-TET1-dCas9 (32 μl). Values and error bars reflect mean ± SD of n = 3 biological replicates. k Representative flow cytometry plots of CLTA-GFP expression in HEK293T cells 15 days post-treatment with RENDER-TET1-dCas9 (32 μl). Data are shown as individual data points and mean ± SD for n = 3 biological replicates. Source data are provided as a Source Data file.

Fig. 2. Optimizing RENDER-CRISPRoff composition.

a Time course of CLTA-GFP silencing in HEK293T cells post-treatment with different doses of RENDER-CRISPRoff (ZIM3) (v1). b Time course of CLTA-GFP silencing in HEK293T cells post-treatment with different doses of RENDER-CRISPRi (ZIM3). In a, b, Values and error bars reflect mean ± SD of n = 3 biological replicates. c, Schematic of optimization of RENDER-CRISPRoff. d Quantification of CLTA-GFP silencing in HEK293T cells 5 days post-treatment with 4 μl RENDER-CRISPRoff with varying gag-CRISPRoff to gag-pol plasmid ratios. X-axis indicates % gag-CRISPRoff plasmid of total amount of gag-CRISPRoff and gag-pol plasmids. Dark gray indicates v1, blue indicates v2. e Quantification of CLTA-GFP silencing in HEK293T cells 5 days post-treatment with 4 μl RENDER-CRISPRoff with varying amounts of sgRNA expressing plasmid. Blue indicates v2, magenta indicates v3. f Dose-response curve of CLTA-GFP silencing in HEK293T cells 5 days post-treatment with RENDER-CRISPRoff v1, v2 and v3. Values and error bars reflect mean ± SD of n = 3 biological replicates. Data were fit to four-parameter logistic curves using nonlinear regression. g Time course of CLTA-GFP silencing in HEK293T cells post-treatment with RENDER-CRISPRoff v3 (16 μl). Values and error bars reflect mean ± SD of n = 3 biological replicates. h Representative histogram plots of CLTA-GFP expression in HEK293T cells at day 3, 7, 14, and 49 post-treatment with RENDER-CRISPRoff v3 (16 μl). UT; untreated. i Representative histogram plots of H2B-GFP expression in HEK293T cells at day 3, 7, 14, and 29 post-treatment with RENDER-CRISPRoff v3 (10 μl). UT; untreated. Source data are provided as a Source Data file.

Fig. 3. Multiplexed epigenetic silencing across diverse human cell types with RENDER-CRISPRoff.

a Quantification of CD55 and CD81 silencing in different cell lines 7 days post-treatment with RENDER-CRISPRoff v3 (8 μl). Data are shown as individual data points and mean ± SD for n = 3 biological replicates. n.e.; not expressed. b Manhattan plot displaying differentially methylated CpGs between Jurkat cells treated with RENDER-CRISPRoff v3 targeting CD55 and untreated cells (14 days post-treatment) analyzed by WGEM-seq. Magenta dots represent CpGs that are more methylated in RENDER-CRISPRoff-treated cells and blue dots represent CpGs that are more methylated in untreated cells. The arrow denotes the genomic location of the CD55 gene. c Plot displaying the % of WGEM-seq reads that are methylated at each CpG across a ± 2 kilobase pairs window from the CD55 transcription start site (TSS) between Jurkat cells treated with RENDER-CRISPRoff v3 targeting CD55 (magenta) or untreated cells (blue) at 14 days posttreatment. Annotated CpG island (CGI) is shown in green and CD55 targeting sgRNA in red. d Volcano plot of RNA-seq data analyses showing differential gene expression between RENDER-CRISPRoff CD55-sgRNAs treated Jurkat cells and untreated Jurkat cells (n = 2). The adjusted p value is a two-sided p value calculated from a DESeq2 Wald test with Benjamini–Hochberg multiple comparisons adjustment. CD55 is shown as a red dot; all other genes are shown as light gray circles. e Volcano plot of RNA-seq data analyses showing differential gene expression between RENDER-CRISPRoff CLTA-sgRNAs treated HEK293T cells and untreated HEK293T cells (n = 2). The adjusted p value is a two-sided p value calculated from a DESeq2 Wald test with Benjamini–Hochberg multiple comparisons adjustment. CLTA is shown as a red dot; all other genes are shown as light gray circles. f Schematic of combinatorial sgRNAs packaging into one RENDER virus-like particle. g Representative flow cytometry plots of CD55 and CD81 expression in Jurkat cells 7 days post-treatment with combinatorial RENDER-CRISPRoff v3 targeting CD55 and CD81 (8 μl). h Representative flow cytometry plots of CD55 and CD81 expression in U-87 MG cells 7 days post-treatment with combinatorial RENDER-CRISPRoff v3 targeting CD55 and CD81 (8 μl). i Quantification of CLTA-GFP silencing in HEK293T cells 3 days post-treatment of 1.0 × 10⁶ cells with different doses of RENDER-CRISPRoff v3. Data are shown as individual data points and mean ± SD for n = 2 biological replicates. j Quantification of CD55 and CD81 silencing in Jurkat cells 3 days post-treatment of 5.0 × 10⁵ cells with different doses of RENDER-CRISPRoff v3. Values represent one biological replicate. k Schematic of sgRNA-free delivery of CRISPRoff apoprotein by RENDER and CLTA-GFP silencing in HEK293T reporter cells stably expressing a CLTA targeting sgRNA. l Dose-response curve of CLTA-GFP silencing in HEK293T cells expressing a CLTA targeting sgRNA 3 days post-treatment with sgRNA-free RENDER-CRISPRoff. Values and error bars reflect mean ± SD of n = 3 biological replicates. Data were fit to four-parameter logistic curves using nonlinear regression. Source data are provided as a Source Data file.

Fig. 4. Epigenome editing in primary human T cells.

a Schematic of RENDER-CRISPRoff treatment and electroporation of isolated primary human T cells. b Representative flow cytometry plot showing the viability of T cells 2 days post RENDER-CRISPRoff v3 or CRISPRoff-mRNA nucleofection with a CD55 targeting sgRNA. c Quantification of live T cells 2 days post RENDER-CRISPRoff v3 or CRISPRoff-mRNA electroporation, with CD55 targeting or non-targeting (NT) sgRNA. The data were normalized to the untreated control group. Data are shown as individual data points and mean ± SD for n = 2 (untreated cells and mRNA (NT) treated cells), or n = 3 (RENDER treated cells and mRNA (CD55) treated cells). Error bars represent the standard deviation. d Representative histogram plots of CD55 expression in T cells at 2 and 11 days post-treatment with RENDER-CRISPRoff v3 (5 μl) targeting CD55 (orange) or non-targeting (gray). Percentages denote % cells with CD55 silenced. e Representative histogram plots of CD55 expression in T cells at 2 and 11 days post-electroporation with CRISPRoff-mRNA and CD55 targeting (orange) or non-targeting (gray) sgRNA. Percentages denote % cells with CD55 silenced. f Quantification of CD55 silencing in primary human T cells 5 days post-treatment with different doses of RENDER-CRISPRoff v3 with CD55-sgRNAs or RENDER-CRISPRoff v3 (4 μl) with non-targeting sgRNA (NT). Data are shown as individual data points and mean ± SD for n = 3 biological replicates. g Quantification of CD81 silencing in primary human T cells 5 days post-treatment with different doses of RENDER-CRISPRoff v3 with CD81-sgRNAs or RENDER-CRISPRoff v3 (4 μl) with non-targeting sgRNA (NT). Data are shown as individual data points and mean ± SD for n = 3 biological replicates. Source data are provided as a Source Data file.

Fig. 5. Epigenome editing iPSC-derived neurons with RENDER.

a Experimental workflow of gene silencing by RENDER-CRISPRoff v3 in iPSC-derived neurons. b Representative histogram plots of CD81 expression in iPSC-derived neurons (wild-type, WT) at day 7 and 14 post-treatment with different doses of RENDER-CRISPRoff v3 targeting CD81 or non-targeting (NT). c Quantification of CD81 expression in iPSC-derived neurons (wild-type, WT) at day 7 and 14 post-treatment with different doses of RENDER-CRISPRoff v3 targeting CD81. CD81 expression is measured by antibody staining and flow cytometry and normalized to non-targeting (NT) control. Data are shown as individual data points and mean ± SD for n = 2 (RENDER-CRISPRoff v3 NT treated neurons), or n = 4 (RENDER-CRISPRoff v3 targeting CD81 treated neurons). Error bars represent the standard deviation. d Representative histogram plots of Tau expression in iPSC-derived neurons (V337M mutant) at day 8 and 15 post-treatment with different doses of RENDER-CRISPRoff v3 targeting MAPT or non-targeting (NT). e Quantification of Tau expression in iPSC-derived neurons (V337M mutant) at day 8 and 15 post-treatment with different doses of RENDER-CRISPRoff v3 targeting MAPT. Tau expression is measured by antibody staining and flow cytometry and normalized to non-targeting (NT) control. Data are shown as individual data points and mean ± SD for n = 2 (RENDER-CRISPRoff v3 NT treated neurons), or n = 4 (RENDER-CRISPRoff v3 targeting MAPT treated neurons). Error bars represent the standard deviation. Source data are provided as a Source Data file.