Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing
Open accessAbstract
A general approach for heritably altering gene expression has the potential to enable many discovery and therapeutic efforts. Here, we present CRISPRoff-a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications. Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons. Pairing CRISPRoff with genome-wide screens and analysis of chromatin marks establishes rules for heritable gene silencing. We identify single guide RNAs (sgRNAs) capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs. The broad ability of CRISPRoff to initiate heritable gene silencing even outside of CGIs expands the canonical model of methylation-based silencing and enables diverse applications including genome-wide screens, multiplexed cell engineering, enhancer silencing, and mechanistic exploration of epigenetic inheritance.
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Written from the paper’s full text.
TL;DR
CRISPRoff is a single dead Cas9 fusion (KRAB–Dnmt3A–Dnmt3L) that transiently writes DNA methylation and repressive histone marks, producing heritable gene silencing through cell division and differentiation. CRISPRon, an optimized TET1-dCas9 plus transactivator system, reverses this silencing, and genome-wide screens show broad targeting of most human genes, including those without canonical CpG islands.
Key findings
- Transient CRISPRoff-V2 silenced Snrpn-GFP in >80% of cells and endogenously tagged HIST2H2BE (H2B) in >90% at 50 days post-transfection; CLTA silencing was maintained in 38 of 39 single-cell clones at 15 months or ~450 cell divisions.
- In genome-wide screens, CRISPRoff produced stronger growth defects than the catalytically inactive Dnmt3AE765A mutant; common essential genes had median γ = −0.2 versus −0.05 for the mutant. Over 300 genes without CGI annotations had growth defects (γ ≤ −0.1), with 160 at γ ≤ −0.2; DYNC2LI1, LAMP2, MYL6, and VPS25 were validated as durably silenced, while CALD1 was not stably silenced at 14 days.
- Active CRISPRoff sgRNAs spanned a broad window around the TSS, notably within ±1 kb, unlike the narrow ~75 bp downstream CRISPRi window; activity was enriched in nucleosome-depleted regions, and H2B-targeting sgRNAs ~2 kb upstream of the TSS were highly active.
- CRISPRon with TETv4 reactivated CLTA in >70% of cells; TETv4 plus Rta or p65-Rta gave higher median reactivation at 28 days. In iPSCs, CD81 silencing persisted in 90% of cells through neuronal differentiation, and MAPT targeting reduced Tau in ~30% of differentiated cells.
Methods
- Transient transfections or nucleofection were performed in HEK293T, HeLa, U2OS, K562, and iPSCs, with FACS enrichment and flow cytometry; silencing was assayed on endogenous GFP-tagged genes and cell-surface proteins, with validation by bisulfite sequencing, WGBS, RNA-seq, and H3K9me3 ChIP-seq.
- Genome-wide pooled CRISPRoff screens used a dual-sgRNA library targeting >20,000 protein-coding genes plus ~1,000 non-targeting controls; tiling libraries covered ±1 kb or ±2.5 kb around TSSs, and phenotypes were calculated as γ from sgRNA enrichment over cell doublings.
- iPSCs were differentiated into neurons using doxycycline-inducible NGN2; PVT1 enhancer silencing was measured by qRT-PCR, and statistical tests and replicate numbers are reported in figure legends.
Limitations
- Stated: most CRISPRoff experiments used sgRNAs previously predicted to be optimal for CRISPRi; experiments were performed in polyclonal cell lines with inherent clonal DNA methylation variation; MYL6 showed metastable silencing, and it remains unknown how many genes are amenable to stable versus metastable silencing.
- Evident from the text: CALD1 was not stably silenced at 14 days, and MYL6 reactivated to near pre-CRISPRoff levels by day 30; modest global DNA methylation increases (<2%) and rare non-target transcript changes were observed, though the authors report these were unlikely to modulate expression or phenotypes.
Model-written summary. Check it against the paper before relying on it.
Integrity signals
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Cites
Statistics
Claims the paper makes
8 of 15 cited sources checked · 3 supported · 1 flagged · accuracy not yet measured
- Premisecitation
Enhancers E1-E4 control expression of the PVT1 long noncoding RNA.
“Finally, we explored the potential utility of CRISPRoff for silencing promoter-distal elements by targeting enhancers that control the expression of the PVT1 long noncoding RNA (Cho et al., 2018; Fulco et al., 2016).”
Quote in the paperSubstantial claim2 of 2 sources checked- cites Cho et al., 2018 source
- cites Fulco et al., 2016 source
The passage describing the seven elements (section labeled Abstract; page unavailable) says e1–e7 are enhancers that control MYC expression, not PVT1 expression. It identifies e3 and e4 as alternative transcription start sites for PVT1, but says they likely affect cellular proliferation through direct regulation of MYC. (LLM, conf=0.88)
Checked by a reasoning model — may be wrong.
Experimental — accuracy not yet measured
- Findingown data
The most active CRISPRoff sgRNAs are located in nucleosome-depleted regions of gene promoters.
“Overlaying nucleosome occupancy with CRISPRoff sgRNA activity scores for all genes showed that the most active sgRNAs are located in nucleosome-depleted regions of gene promoters, as we and others have shown previously for Cas9 and dCas9-based tools (Figures 6J, 6K, S6D and S6E) (Horlbeck et al., 2016b; Isaac et al., 2016).”
Quote in the paperSubstantial claim1 of 2 sources checked- cites Horlbeck et al., 2016b
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Isaac et al., 2016 source
- Premisecitation
MAPT expression increases substantially during neuronal differentiation.
“MAPT is transcriptionally repressed in iPSCs by H3K27me3 rather than by DNA methylation and H3K9me3 and its expression increases substantially during neuronal differentiation (Guenther et al., 2010).”
Quote in the paperSubstantial claim1 of 1 sources checked- cites Guenther et al., 2010 source
The Introduction and Discussion describe chromatin marks and gene-expression comparisons between ESCs and iPSCs, but do not report MAPT expression during neuronal differentiation. Searches for MAPT, tau, and neuronal differentiation found no passage addressing the claimed increase. (LLM, conf=0.76)
Experimental — accuracy not yet measured
- Findingown data
CRISPRoff can perturb enhancers.
“Our initial experiments demonstrate CRISPRoff can perturb enhancers, opening the potential to target genome elements that control tissue-specific gene expression (Fulco et al., 2016; Tarjan et al., 2019).”
Quote in the paperNot a substantial claim2 of 2 sources checked- cites Fulco et al., 2016 source
The paper reports that targeting enhancers with CRISPR interference (CRISPRi) reduced GATA1 expression, supporting the general point that enhancers can be perturbed. It does not report experiments with CRISPRoff, so it does not establish that CRISPRoff specifically can perturb enhancers (results described under the Abstract section; page not supplied). (LLM, conf=0.88)
Experimental — accuracy not yet measured - cites Tarjan et al., 2019 source
In Results, under “Epigenome editing can disrupt CTCF at topological insulators” (page unavailable), the paper says dCas9-KRAB can repress enhancer function. Its experiments, however, target CTCF insulators, and our copy does not identify or test CRISPRoff as an enhancer-perturbing method. (LLM, conf=0.83)
Experimental — accuracy not yet measured
- Study design and conductcitation
Most CRISPRoff experiments, including genome-wide screens, were performed with sgRNAs previously predicted to be optimal for CRISPRi.
“Most of our CRISPRoff experiments, including the genome-wide screens, were performed with sgRNAs previously predicted to be optimal for CRISPRi (Horlbeck et al., 2016a; Replogle et al., 2020).”
Quote in the paperNot a substantial claim1 of 2 sources checked- cites Horlbeck et al., 2016a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
- cites Replogle et al., 2020 source
- Findingown data
For the 39 genes without promoter CGIs, many highly active sgRNAs have phenotype strength comparable to genes with annotated CGIs.
“Analysis of the 39 genes without promoter CGIs showed many highly active sgRNAs of comparable phenotype strength to genes with annotated CGIs (Figure 6C, colored red) and the phenotypes were strongly diminished in the CRISPRoff mutant screen.”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Findingown data
Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons.
“Transient CRISPRoff expression initiates highly specific DNA methylation and gene repression that is maintained through cell division and differentiation of stem cells to neurons.”
Quote in the paperSubstantial claim - Findingown data
The RAB11A promoter is nucleosome-dense.
“Overlaying nucleosome occupancy with sgRNA activity showed that the RAB11A promoter is nucleosome-dense (Figure S6H).”
Quote in the paperSubstantial claim - Findingown data
CD81-off neurons had heavily methylated promoter CpG dinucleotides compared to neurons treated with CRISPRoff and a non-targeting sgRNA.
“We harvested genomic DNA from CD81-off neurons and detected heavily methylated promoter CpG dinucleotides compared to neurons treated with CRISPRoff and a non-targeting sgRNA (Figure 7E).”
Quote in the paperSubstantial claim - Findingown data
Genome-wide CRISPRoff screens show that CRISPRoff can durably and specifically silence the large majority of protein-coding genes.
“Using genome-wide CRISPRoff screens, we show that this approach can durably and specifically silence the large majority of protein-coding genes and has a wide targeting window across gene promoters.”
Quote in the paperSubstantial claim - Findingown data
At 30 days post-transfection, many iPSCs had stably silenced CD81 after CRISPRoff and CD81-targeting sgRNAs.
“We transfected iPSCs with CRISPRoff and sgRNAs targeting CD81 or a non-targeting control and found that at 30 days post-transfection, many iPSCs had stably silenced CD81 (Figures 7A and 7B).”
Quote in the paperSubstantial claim - Findingown data
sgRNAs targeting ~2 kb upstream of the H2B TSS were highly active.
“Unexpectedly, sgRNAs targeting ~2 kb upstream of the H2B TSS were highly active (Figure 6L).”
Quote in the paperNot a substantial claim - Findingown data
A high degree of CpG methylation was detected between the sgRNA binding sites.
“We also detected a high degree of CpG methylation between the sgRNA binding sites, suggesting a linear movement of spreading from the site of initiation (site 2, Figure S7I).”
Quote in the paperSubstantial claim - Findingown data
For the 39 genes without annotated CGIs, CRISPRoff has a broadened targeting window.
“Moreover, we observed that CRISPRoff has a broadened targeting window despite the lack of an annotated CGI for these 39 genes (Figure 6I).”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Findingown data
At day 10 of differentiation, ~30% of cells had reduced Tau expression compared to a non-targeting control after CRISPRoff and MAPT-targeting sgRNAs.
“At day 10 of the differentiation protocol, we measured Tau protein levels and found ~30% of cells with reduced Tau expression compared to a non-targeting control (Figures 7F-H).”
Quote in the paperSubstantial claim - Findingown data
CRISPRoff has a wide targeting window across gene promoters, extending beyond annotated CGIs.
“We identify sgRNAs capable of silencing the large majority of genes including those lacking canonical CpG islands (CGIs) and reveal a wide targeting window extending beyond annotated CGIs.”
Quote in the paperSubstantial claim - Findingown data
Active CRISPRoff sgRNAs are not evenly distributed but appear in a periodic pattern within the -500 to +500 bp window, as shown for DKC1, GPN2, and ZCCHC9.
“We observed that active sgRNAs are not evenly distributed but instead appear in a periodic pattern within the –500 to +500 bp window, as shown for DKC1, GPN2, and ZCCHC9 (Figures 6E–6G).”
Quote in the paperNot a substantial claim - Findingown data
At day 5, CpG methylation was established and spread from the site of initiation to a ~2 kb site from the sgRNA binding site.
“At day 5, we detected establishment and spreading of CpG methylation from the site of initiation to a ~2 kb site from the sgRNA binding site (labeled site 1 and site 3, Figures S7E and S7F).”
Quote in the paperSubstantial claim - Findingown data
The phenotypes of highly active sgRNAs for the 39 genes without promoter CGIs were strongly diminished in the CRISPRoff mutant screen.
“Analysis of the 39 genes without promoter CGIs showed many highly active sgRNAs of comparable phenotype strength to genes with annotated CGIs (Figure 6C, colored red) and the phenotypes were strongly diminished in the CRISPRoff mutant screen.”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Findingown data
Active sgRNAs for CLTA, H2B, and VIM spanned a large window across the TSS.
“Similar to the growth screens, active sgRNAs for CLTA, H2B, and VIM spanned a large window across the TSS (Figures 6L and S6G–S6I).”
Quote in the paperSubstantial claim - Findingown data
For VIM, active sgRNAs spanned a 2 kb window +/− 1 kb from the TSS.
“Similarly, for VIM, active sgRNAs spanned a 2 kb window +/− 1 kb from the TSS.”
Quote in the paperNot a substantial claim - Findingown data
Acquired H3K9me3 modifications in CRISPRoff-treated cells overlapped with the unmethylated CpG region in untreated parental cells.
“The acquired H3K9me3 modifications in CRISPRoff-treated cells overlapped with the unmethylated CpG region in untreated parental cells (bottom track in Figure S7C).”
Quote in the paperNot a substantial claim - Findingown data
A similar fraction of undifferentiated iPSCs maintained CD81 silencing during the same time course.
“A similar fraction of undifferentiated iPSCs maintained CD81 silencing during the same time course, suggesting that the reactivation of CD81 in ~10% of cells was not due to the differentiation process.”
Quote in the paperSubstantial claim - Findingown data
Canonical CpG island annotations are not necessary for stable gene silencing by CRISPRoff.
“Surprisingly, canonical CpG island annotations are not necessary for stable gene silencing by CRISPRoff.”
Quote in the paperSubstantial claim - Findingnone given
CRISPRoff epigenetic memories can be reversed robustly using CRISPRon, which removes DNA methylation and recruits transcriptional machinery.
“CRISPRoff epigenetic memories can be reversed robustly using a multi-partite epigenetic editor we call CRISPRon, which removes DNA methylation and recruits transcriptional machinery.”
Quote in the paperSubstantial claim - Findingown data
By day 30, DNA methylation was stably maintained at both sgRNA binding sites.
“By day 30, we detected stable maintenance of DNA methylation at both sgRNA binding sites (Figures S7G and S7H).”
Quote in the paperSubstantial claim- Rests on an assumption: Polyclonal cell lines inherently have clonal variations in DNA methylation.
- Findingown data
Active sgRNAs for RAB11A were constricted to a narrow window at the TSS.
“By contrast, active sgRNAs for RAB11A were constricted to a narrow window at the TSS.”
Quote in the paperSubstantial claim - Findingown data
For ORC5, active CRISPRoff sgRNAs are spread across −500 to +500 bp from the TSS, similarly to genes with annotated CGIs.
“A representative gene plot of ORC5 shows that similarly to genes with annotated CGIs, active CRISPRoff sgRNAs are spread across −500 to +500 bp from the TSS (Figure 6H).”
Quote in the paperSubstantial claim - Findingown data
At 30 days post CRISPRoff transfection, 89% of cells maintained H2B silencing when sgRNA-A was delivered compared to 76% with sgRNA-B.
“At 30 days post CRISPRoff transfection, 89% of cells maintained H2B silencing when sgRNA-A was delivered compared to 76% with sgRNA-B (Figure S7B).”
Quote in the paperSubstantial claim - Findingown data
CD81 remained silenced during and after neuronal differentiation in 90% of cells.
“We observed that CD81 remained silenced during and after neuronal differentiation in 90% of cells (Figures 7C and 7D).”
Quote in the paperSubstantial claim - Findingnone given
CRISPRoff is a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications.
“Here, we present CRISPRoff—a programmable epigenetic memory writer consisting of a single dead Cas9 fusion protein that establishes DNA methylation and repressive histone modifications.”
Quote in the paperSubstantial claim - Findingown data
Deposition and maintenance of H3K9me3 was far weaker with CRISPRoff bearing the D3A mutation.
“In contrast, deposition and maintenance of H3K9me3 was far weaker with CRISPRoff bearing the D3A mutation, consistent with the failure to sustain gene repression with the mutant (Figure S7D).”
Quote in the paperSubstantial claim - Findingown data
At 4 weeks after CRISPRoff transfection in the RAB11A GFP-tagged HEK293T tiling screen, the detectable GFP-silenced population was 3.05%.
“At the 4 weeks time point, each cell line had the following detectable GFP-silenced population: 21.8% CLTA, 22.7% HIST2H2BE, 3.05% RAB11A, and 24.7% VIM.”
Quote in the paperNot a substantial claim - Findingown data
At 4 weeks after CRISPRoff transfection in the HIST2H2BE GFP-tagged HEK293T tiling screen, the detectable GFP-silenced population was 22.7%.
“At the 4 weeks time point, each cell line had the following detectable GFP-silenced population: 21.8% CLTA, 22.7% HIST2H2BE, 3.05% RAB11A, and 24.7% VIM.”
Quote in the paperNot a substantial claim - Findingown data
CRISPRoff-Dnmt3AE765A mutant resulted in less robust PVT1 knockdown than CRISPRoff.
“In contrast, parallel experiments using the CRISPRoff-Dnmt3AE765A mutant resulted in less robust knockdown (Figure 7J, right).”
Quote in the paperNot a substantial claim - Findingown data
Targeted DNA methylation outside of annotated CGIs can lead to robust memorized gene silencing.
“Our finding that targeted DNA methylation outside of annotated CGIs can lead to robust memorized gene silencing extends the canonical model of methylation-based gene silencing, which posits that a high density of CpG methylation is a requirement for stable propagation of silencing (Boyes and Bird, 1992).”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Rests on an assumption: Polyclonal cell lines inherently have clonal variations in DNA methylation.
- Findingown data
Targeting CRISPRoff 2 kb upstream of the H2B TSS leads to acquisition and maintenance of H3K9me3 and DNA methylation marks at the same genomic positions as targeting CRISPRoff directly proximal to the TSS.
“For example, targeting CRISPRoff 2 kb upstream of the H2B TSS leads to acquisition and maintenance of H3K9me3 and DNA methylation marks at the same genomic positions as targeting CRISPRoff directly proximal to the TSS, pointing to the existence of preexisting boundaries that restrict epigenetic spreading.”
Quote in the paperSubstantial claim - Findingown data
At 4 weeks after CRISPRoff transfection in the CLTA GFP-tagged HEK293T tiling screen, the detectable GFP-silenced population was 21.8%.
“At the 4 weeks time point, each cell line had the following detectable GFP-silenced population: 21.8% CLTA, 22.7% HIST2H2BE, 3.05% RAB11A, and 24.7% VIM.”
Quote in the paperNot a substantial claim - Findingown data
No transcriptional knockdown of BOLA1 was detected in RNA-seq data.
“We do not detect transcriptional knockdown of BOLA1 in our RNA-seq data (Table S1).”
Quote in the paperNot a substantial claim - Findingown data
At 4 weeks after CRISPRoff transfection in the VIM GFP-tagged HEK293T tiling screen, the detectable GFP-silenced population was 24.7%.
“At the 4 weeks time point, each cell line had the following detectable GFP-silenced population: 21.8% CLTA, 22.7% HIST2H2BE, 3.05% RAB11A, and 24.7% VIM.”
Quote in the paperNot a substantial claim - Findingown data
sgRNAs targeting E1, E3, and E4 significantly reduced PVT1 transcript levels by ~40-60%, compared to 80% knockdown with promoter-targeting sgRNAs.
“We detected a significant reduction of PVT1 transcript levels with sgRNAs targeting E1, E3, and E4 (~40–60%) compared to 80% knockdown with promoter-targeting sgRNAs (Figure 7J, left).”
Quote in the paperSubstantial claim - Interpretationown data
CRISPRoff-mediated epigenome editing is an applicable technology for rewriting gene expression programs in iPSC-derived cells, especially for modulating gene expression in cells where delivery of gene editing platforms remains a challenge.
“Together, these data support CRISPRoff-mediated epigenome editing as an applicable technology for rewriting gene expression programs in iPSC-derived cells, especially for modulating gene expression in cells where delivery of gene editing platforms remains a challenge.”
Quote in the paperSubstantial claim - Interpretationreasoning
Reactivation of CD81 in ~10% of cells was not due to the differentiation process.
“A similar fraction of undifferentiated iPSCs maintained CD81 silencing during the same time course, suggesting that the reactivation of CD81 in ~10% of cells was not due to the differentiation process.”
Quote in the paperSubstantial claim - Interpretationown data
CRISPRoff-encoded memory of silencing is stably maintained in stem cells.
“Thus, CRISPRoff-encoded memory of silencing is stably maintained in stem cells.”
Quote in the paperSubstantial claim - Interpretationown data
CRISPRoff accessibility is restricted by nucleosomes.
“From these data, we interpret that CRISPRoff accessibility is restricted by nucleosomes; however, once bound, CRISPRoff can silence gene expression even when distal to the TSS.”
Quote in the paperSubstantial claim - Interpretationown data
Histone and DNA methylation deposition, spreading, and maintenance are orchestrated.
“These data, together with our WGBS data, highlight the orchestration of histone and DNA methylation deposition, spreading, and maintenance and suggest that there are underlying regulatory principles that likely depend on the genomic context.”
Quote in the paperSubstantial claim - Interpretationown data
The optimal window for CRISPRoff gene silencing is similarly broad for genes with and without annotated CGIs.
“Our experiments demonstrate that the optimal window for CRISPRoff gene silencing is similarly broad for genes with and without annotated CGIs, likely due to low density CpG sites that are functional for methylation-dependent gene silencing as we demonstrated for DYNC2LI1, LAMP2, MYL6, and VPS25 (Figure 5C).”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Interpretationown data
Once bound, CRISPRoff can silence gene expression even when distal to the TSS.
“From these data, we interpret that CRISPRoff accessibility is restricted by nucleosomes; however, once bound, CRISPRoff can silence gene expression even when distal to the TSS.”
Quote in the paperSubstantial claim - Interpretationown data
The weaker H3K9me3 deposition and maintenance with the D3A mutant is consistent with the failure to sustain gene repression with the mutant.
“In contrast, deposition and maintenance of H3K9me3 was far weaker with CRISPRoff bearing the D3A mutation, consistent with the failure to sustain gene repression with the mutant (Figure S7D).”
Quote in the paperSubstantial claim - Interpretationown data
Depletion of DNA methylation is sufficient to reverse CRISPRoff-mediated gene silencing.
“These results demonstrate that depletion of DNA methylation is sufficient to reverse CRISPRoff-mediated gene silencing and motivated us to optimize programmable tools for reactivation of CRISPRoff-silenced genes.”
Quote in the paperSubstantial claim - Interpretationown data
Underlying regulatory principles of histone and DNA methylation deposition, spreading, and maintenance likely depend on genomic context.
“These data, together with our WGBS data, highlight the orchestration of histone and DNA methylation deposition, spreading, and maintenance and suggest that there are underlying regulatory principles that likely depend on the genomic context.”
Quote in the paperSubstantial claim - Interpretationown data
CpG methylation spreads by linear movement from the site of initiation.
“We also detected a high degree of CpG methylation between the sgRNA binding sites, suggesting a linear movement of spreading from the site of initiation (site 2, Figure S7I).”
Quote in the paperNot a substantial claim - Interpretationnone given
The CRISPRoff-written gene silencing program can be rapidly reversed by CRISPRon.
“Transient expression of CRISPRoff writes a robust, specific, and multiplexable gene silencing program that is memorized by cells through cell division and differentiation, which can be rapidly reversed by CRISPRon.”
Quote in the paperSubstantial claim - Interpretationown data
CRISPRoff can specifically and robustly silence the large majority of human genes.
“We show that CRISPRoff can specifically and robustly silence the large majority of human genes.”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
- Interpretationreasoning
The difference in phenotype scores between K562 CRISPRi and HEK293T CRISPRoff screens likely can be attributed to cell type variability between K562 and HEK293T, because the genes were chosen based on essentiality in K562s.
“However, as we have previously demonstrated, because the genes were chosen based on essentiality in K562s, this difference likely can be attributed to cell type variability between K562 and HEK293T.”
Quote in the paperNot a substantial claim - Interpretationnone given
Transient expression of CRISPRoff writes a robust, specific, and multiplexable gene silencing program.
“Transient expression of CRISPRoff writes a robust, specific, and multiplexable gene silencing program that is memorized by cells through cell division and differentiation, which can be rapidly reversed by CRISPRon.”
Quote in the paperSubstantial claim - Interpretationnone given
CRISPRoff and CRISPRon are technologies for programmably writing and erasing epigenetic memories to control gene expression programs.
“Here, we present CRISPRoff and CRISPRon, two technologies for programmably writing and erasing epigenetic memories to control gene expression programs.”
Quote in the paperSubstantial claim - Interpretationown data
The CRISPRoff-written gene silencing program is memorized by cells through cell division and differentiation.
“Transient expression of CRISPRoff writes a robust, specific, and multiplexable gene silencing program that is memorized by cells through cell division and differentiation, which can be rapidly reversed by CRISPRon.”
Quote in the paperSubstantial claim - Interpretationown data
Preexisting boundaries restrict epigenetic spreading.
“For example, targeting CRISPRoff 2 kb upstream of the H2B TSS leads to acquisition and maintenance of H3K9me3 and DNA methylation marks at the same genomic positions as targeting CRISPRoff directly proximal to the TSS, pointing to the existence of preexisting boundaries that restrict epigenetic spreading.”
Quote in the paperSubstantial claim - Interpretationown data
The finding that targeted DNA methylation outside of annotated CGIs can lead to robust memorized gene silencing extends the canonical model.
“Our finding that targeted DNA methylation outside of annotated CGIs can lead to robust memorized gene silencing extends the canonical model of methylation-based gene silencing, which posits that a high density of CpG methylation is a requirement for stable propagation of silencing (Boyes and Bird, 1992).”
Quote in the paperSubstantial claim
- Classified as probably not a substantial claim. 19
- May combine two claims. 13
- The quote only partly states this claim. 2
Probably not a substantial claim: may not pass S3 (load-bearing).
PremisecitationMAPT is transcriptionally repressed in iPSCs by H3K27me3 rather than by DNA methylation and H3K9me3.
“MAPT is transcriptionally repressed in iPSCs by H3K27me3 rather than by DNA methylation and H3K9me3 and its expression increases substantially during neuronal differentiation (Guenther et al., 2010).”
Quote in the paperNot a substantial claim1 of 1 sources checked- cites Guenther et al., 2010 source
The Results and Discussion section reports that H3K27me3 occurs at promoters of many repressed genes in iPSCs, but does not identify MAPT among them. Searches for MAPT and for the proposed comparison with DNA methylation and H3K9me3 found no passage establishing how MAPT is repressed; the copy also does not establish its expression change during neuronal differentiation. Page numbers were not available in the indexed passages. (LLM, conf=0.76)
Experimental — accuracy not yet measured
May combine two claims.
Findingown dataBoth sgRNA-A and sgRNA-B induced deposition at day 5 and maintenance at day 30 of H3K9me3 across the H2B locus despite the ~2 kb distance between the sgRNA binding sites.
“Using ChIP-seq, we showed that both sgRNAs induced deposition at day 5 and maintenance at day 30 of H3K9me3 across the locus despite the ~2 kb distance between the sgRNA binding sites (Figure S7C).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataActive CRISPRoff sgRNAs for CLTA were within two distinct regions, with one region upstream of the TSS outside of the annotated CGI.
“Active CRISPRoff sgRNAs for CLTA were within two distinct regions, with one region upstream of the TSS outside of the annotated CGI (Figure S6G).”
Quote in the paperNot a substantial claimThe quote only partly states this claim.
Findingown dataActive CRISPRoff sgRNAs are broadly distributed across the TSS, notably within a 1 kb window centered on the TSS, unlike CRISPRi.
“In contrast, the active CRISPRoff sgRNAs were broadly distributed across the TSS, notably within a 1 kb window centered on the TSS.”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataCRISPRoff can durably silence gene expression, with memory maintained for more than 450 cell divisions.
“Transient expression of CRISPRoff writes an epigenetic program that human cells maintain for more than 450 cell divisions, highlighting that this form of gene silencing is stable and heritable.”
Quote in the paperSubstantial claimMay combine two claims. The quote only partly states this claim.
Findingown dataCRISPRoff silencing is highly specific, with minimal off-target gene knockdown and confined DNA methylation.
“Comparison of untransfected cells with cells transfected with CRISPRoff show minimal off-target gene knockdown (Figure 2A).”
Quote in the paperSubstantial claimMay combine two claims.
Findingown dataChIP-seq data show enrichment for H3K9me3 near BOLA1 TSS1, the closest to the H2B promoter, and no enrichment for TSS2 located 15 kb away.
“We note that BOLA1 contains two TSS annotations and our ChIP-seq data show enrichment for H3K9me3 near TSS1, the closest to the H2B promoter, and no enrichment for TSS2 located 15 kb away (Figures 2G and 2H).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Findingnone givenNon-target differentially expressed transcripts are lowly expressed genes.
“We note that non-target differentially expressed transcripts are lowly expressed genes.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingown dataIn K562 CRISPRi screens, the median sgRNA phenotype score (γ) was −0.46, which is more pronounced than the median γ of −0.33 in the HEK293T CRISPRoff screen.
“We note that the phenotypes in K562 CRISPRi (median γ = −0.46) are more pronounced compared to the HEK293T CRISPRoff screen (median γ = −0.33).”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Findingnone givenMYL6 is a non-CGI gene that has metastable silencing over time.
“Lastly, we highlight MYL6 as a non-CGI gene that has metastable silencing over time.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
PremisecitationMAPT encodes the Tau protein and is implicated in various neurological diseases.
“We next applied CRISPRoff-mediated editing of iPSC-derived neurons to silence MAPT, a gene that encodes the Tau protein and is implicated in various neurological diseases (Iqbal et al., 2016).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Iqbal et al., 2016 source
May combine two claims.
Interpretationown dataThe CRISPRoff screen resulted in high positive rate of calling essential genes with low false positive gene hits, suggesting that CRISPRoff has the ability to silence the majority of genes in the human genome.
“Collectively, the CRISPRoff screen resulted in high positive rate of calling essential genes with low false positive gene hits, suggesting that CRISPRoff has the ability to silence the majority of genes in the human genome (Figure 4F).”
Quote in the paperSubstantial claimMay combine two claims.
Interpretationown dataThe broad optimal window for CRISPRoff gene silencing is likely due to low density CpG sites that are functional for methylation-dependent gene silencing, as demonstrated for DYNC2LI1, LAMP2, MYL6, and VPS25.
“Our experiments demonstrate that the optimal window for CRISPRoff gene silencing is similarly broad for genes with and without annotated CGIs, likely due to low density CpG sites that are functional for methylation-dependent gene silencing as we demonstrated for DYNC2LI1, LAMP2, MYL6, and VPS25 (Figure 5C).”
Quote in the paperSubstantial claim- Rests on an assumption: The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
May combine two claims.
Interpretationown dataEpigenetic editing using CRISPRoff is not limited to genes with canonical CGI annotations and can be targeted to most genes encoded in the human genome.
“Together, these data establish that epigenetic editing using CRISPRoff is not limited to genes with canonical CGI annotations and can be targeted to most genes encoded in the human genome.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataOver 98% of sequencing reads were aligned as proper pairs across samples.
“Over 98% of reads were aligned as proper pairs across samples.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
Study design and conductown dataEach sgRNA was represented by at least 1000 cells throughout the duration of the screens.
“The screens were performed with two technical replicates and each sgRNA was represented by at least 1000 cells throughout the duration of the screens.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenFor the most stringent threshold, sgRNAs were required to have no more than 1 alignment (the sgRNA target site itself) in the genome with a mismatch score of 39.
“For the most stringent threshold, sgRNAs were required to have no more than 1 alignment (the sgRNA target site itself) in the genome with a mismatch score of 39.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationsgRNAs were scored for off-target sites using weighted Bowtie, as previously described.
“The sgRNAs were scored for off-target sites using weighted Bowtie, as previously described (Horlbeck et al., 2016a).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Horlbeck et al., 2016a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductnone givenThe previously published endogenously GFP-tagged cell lines (CLTA, HIST2H2BE, RAB11A, VIM) were further FACS sorted to yield >99% GFP-positive cells to minimize background GFP-negative cells.
“The previously published endogenously GFP-tagged cell lines (CLTA, HIST2H2BE, RAB11A, VIM) were further FACS sorted to yield >99% GFP-positive cells to minimize background GFP-negative cells.”
Quote in the paperNot a substantial claimMay combine two claims.
InterpretationcitationCRISPRoff gene silencing can lead to effectively complete nulls without inducing a DNA damage response.
“CRISPRoff gene silencing can lead to effectively complete nulls without inducing a DNA damage response facilitating multigene targeting screens or therapeutic cell engineering (Ihry et al., 2018).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Ihry et al., 2018 source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataBisulfite conversion efficiency was estimated to be greater than 99.5% based on non-CpG methylation.
“Bisulfite conversion efficiency was estimated to be greater than 99.5% based on non-CpG methylation.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductreasoningTrimming is required to remove bases added during the Adaptase reaction that could affect alignment and DNA methylation calling.
“Trimming is required to remove bases that are added during the Adaptase reaction that could affect alignment and DNA methylation calling.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataCRISPRoff experiments were performed in polyclonal cell lines.
“Moreover, we performed our experiments in polyclonal cell lines, which inherently have clonal variations in DNA methylation.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataAverage CpG coverage was approximately 25-fold across samples.
“Average CpG coverage was ~25-fold across samples.”
Quote in the paperNot a substantial claimClassified as probably not a substantial claim.
PremisecitationThe canonical model of methylation-based gene silencing posits that high density of CpG methylation is required for stable propagation of silencing.
“Our finding that targeted DNA methylation outside of annotated CGIs can lead to robust memorized gene silencing extends the canonical model of methylation-based gene silencing, which posits that a high density of CpG methylation is a requirement for stable propagation of silencing (Boyes and Bird, 1992).”
Quote in the paperSubstantial claim0 of 1 sources checked- cites Boyes and Bird, 1992 source
Probably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductcitationControl non-targeting sgRNAs were extracted from a previously tested list of control sgRNAs.
“Control non-targeting sgRNAs were extracted from a previously tested list of control sgRNAs (Horlbeck et al., 2016a).”
Quote in the paperNot a substantial claim0 of 1 sources checked- cites Horlbeck et al., 2016a
Couldn’t be matched to a paper in Crossref, so it can’t be checked.
Classified as probably not a substantial claim.
Study design and conductnone givenExperiments comparing silencing activity of different CRISPRoff fusions were performed in cells stably expressing the targeting sgRNA to normalize sgRNA expression.
“Experiments that compare the silencing activity of different CRISPRoff fusions (Figures 1E, 4C, S1B, and S1F) were performed in cells that stably express the targeting sgRNA to normalize sgRNA expression.”
Quote in the paperSubstantial claimProbably not a substantial claim: may not pass S3 (load-bearing).
Study design and conductown dataThe screens were performed with two technical replicates.
“The screens were performed with two technical replicates and each sgRNA was represented by at least 1000 cells throughout the duration of the screens.”
Quote in the paperNot a substantial claimProbably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.
Study design and conductown dataCell viability was severely reduced past 9 days post sgRNA infection, so the last time point was taken at 9 days.
“The last time point was taken at 9 days post sgRNA infection, as cell viability was severely reduced past this time point.”
Quote in the paperNot a substantial claim
The paper relies on this without support.
The Dnmt3AE765A mutant is catalytically inactive and unable to maintain durable gene silencing, so it mirrors transient CRISPRi.
“As a control, we performed in parallel an identical screen with a CRISPRoff variant encoding the Dnmt3AE765A mutation, which is catalytically inactive and thus unable to maintain durable gene silencing and instead mirrors the transient silencing effect of CRISPRi (Figure 4C).”
The paper relies on this without support.
The sgRNAs previously predicted to be optimal for CRISPRi are adequate for CRISPRoff experiments.
“Most of our CRISPRoff experiments, including the genome-wide screens, were performed with sgRNAs previously predicted to be optimal for CRISPRi (Horlbeck et al., 2016a; Replogle et al., 2020).”
The paper relies on this without support.
Polyclonal cell lines inherently have clonal variations in DNA methylation.
“Moreover, we performed our experiments in polyclonal cell lines, which inherently have clonal variations in DNA methylation.”
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
A new set of epigenetic editor CRISPRoff has been developed, enabling persistent, heritable, and reversible DNA methylation modification and regulation of gene transcription.
“Recently, a new set of epigenetic editor CRISPRoff has been developed, enabling persistent, heritable, and reversible DNA methylation modification and regulation of gene transcription.430”
From Epigenetic regulation in major depression and other stress-related disorders: molecular mechanisms, clinical relevance and therapeutic potential (2023) · cites this paper as [430]
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Mapping information-rich genotype-phenotype landscapes with genome-scale Perturb-seq
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MethodSelf-citationPast, present, and future of CRISPR genome editing technologies
Pacesa, M., Pelea, O., & Jinek, M. (2024) · Cell · doi:10.1016/j.cell.2024.01.042 · Cited by 609 · see citing works
Hsieh, T.-H. S., Cattoglio, C., Slobodyanyuk, E., Hansen, A. S., Darzacq, X., & Tjian, R. (2022) · Nature Genetics · doi:10.1038/s41588-022-01223-8 · Cited by 406 · see citing works
Delivery of CRISPR-Cas tools for in vivo genome editing therapy: Trends and challenges
Taha, E. A., Lee, J., & Hotta, A. (2022) · Journal of Controlled Release · doi:10.1016/j.jconrel.2022.01.013 · Cited by 259 · see citing works
Yuan, M., Yang, B., Rothschild, G., Mann, J. J., Sanford, L. D., Tang, X., Huang, C., Wang, C., & Zhang, W. (2023) · Signal Transduction and Targeted Therapy · doi:10.1038/s41392-023-01519-z · Cited by 246 · see citing works
Genotoxic effects of base and prime editing in human hematopoietic stem cells
Fiumara, M., Ferrari, S., Omer-Javed, A., Beretta, S., Albano, L., Canarutto, D., Varesi, A., Gaddoni, C., Brombin, C., Cugnata, F., et al. (2024) · Nature Biotechnology · doi:10.1038/s41587-023-01915-4 · Cited by 220 · see citing works
A functional genetic toolbox for human tissue-derived organoids
Sun, D., Evans, L., Perrone, F., Sokleva, V., Lim, K., Rezakhani, S., Lutolf, M., Zilbauer, M., & Rawlins, E. L. (2021) · eLife · doi:10.7554/elife.67886 · Cited by 71 · see citing works
Tycko, J., Van, M. V., Aradhana, DelRosso, N., Ye, H., Yao, D., Valbuena, R., Vaughan-Jackson, A., Xu, X., Ludwig, C., et al. (2025) · Nature Biotechnology · doi:10.1038/s41587-024-02442-6 · Cited by 57 · see citing works
PICALM Alzheimer’s risk allele causes aberrant lipid droplets in microglia
Kozlova, A., Zhang, S., Sudwarts, A., Zhang, H., Smirnou, S., Byeon, S. K., Thapa, C., Sun, X., Stephenson, K., Zhao, X., et al. (2025) · Nature · doi:10.1038/s41586-025-09486-x · Cited by 53 · see citing works
“The gRNA sequences were designed using Benchling (https://benchling.com; Supplementary Table 19), and cloned into the CROPseq-Guide-Puro vector (Addgene, 86708) for co-expression with CRISPRoff v.2.1 (Addgene, 167981) as described previously 77 .”
InfluentialEmerging applications of gene editing technologies for the development of climate-resilient crops
Chavhan, R. L., Jaybhaye, S. G., Hinge, V. R., Deshmukh, A. S., Shaikh, U. S., Jadhav, P. K., Kadam, U. S., & Hong, J. C. (2025) · Frontiers in Genome Editing · doi:10.3389/fgeed.2025.1524767 · Cited by 43 · see citing works
Analog epigenetic memory revealed by targeted chromatin editing
Palacios, S., Bruno, S., Weiss, R., Salibi, E., Kane, A., Ilia, K., & Del Vecchio, D. (2024) · doi:10.1101/2024.02.13.580200 · Cited by 13 · see citing works
Neurogenesis and Neuroinflammation in Dialogue: Mapping Gaps, Modulating Microglia, Rewiring Aging
Tanaka, M. (2026) · Cells · doi:10.3390/cells15010078 · Cited by 11 · see citing works
- Authors
- Nuñez, J. K., Chen, J., Pommier, G. C., Cogan, J. Z., Replogle, J. M., Adriaens, C., Ramadoss, G. N., Shi, Q., Hung, K. L., Samelson, A. J., Pogson, A. N., Kim, J. Y. S., Chung, A., Leonetti, M. D., Chang, H. Y., Kampmann, M., Bernstein, B. E., Hovestadt, V., Gilbert, L. A., & Weissman, J. S.
- Affiliations
- Howard Hughes Medical Institute (US)
- University of California, San Francisco (US)
- Broad Institute (US)
- Stanford University (US)
- Chan Zuckerberg Biohub San Francisco (US)
- Corresponding author
- Gilbert, Luke A.
- Weissman, Jonathan S.
- ORCID
- Venue
- Cell
- Publisher
- Elsevier BV
- Published
- 2021-04
- Volume
- 184
- Issue
- 9
- Pages
- 2503-2519.e17
- ISSN
- 0092-8674 (ISSN-L)
- PMC
- PMC8376083
- OpenAlex
- W3141751158
- Semantic Scholar
- 7ab01c80a6442103e1226c286af71577733cd31a
- Cited by
- 760
- References
- 64
- Citations by year
- 2026: 153 · 2025: 161 · 2024: 162 · 2023: 113 · 2022: 120 · 2021: 50
- FWCI
- 35.98
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.elsevier.com/tdm/userlicense/1.0/ (tdm)
- www.elsevier.com/legal/tdmrep-license (tdm)
- www.elsevier.com/open-access/userlicense/1.0/ (vor, 393 d)
- doi.org/10.15223/policy-017 (stm-asf)
- doi.org/10.15223/policy-037 (stm-asf)
- doi.org/10.15223/policy-012 (stm-asf)
- doi.org/10.15223/policy-029 (stm-asf)
- doi.org/10.15223/policy-004 (stm-asf)
- Locations
- Cell (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- DSpace@MIT (Massachusetts Institute of Technology) (submittedVersion, cc-by-nc-nd)
- eScholarship (California Digital Library) (submittedVersion)
- Funders
- NIH
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Keywords
- epigenome editing, DNA methylation, single guide RNA, CpG islands, histone modifications, epigenetic inheritance, enhancer silencing, genome-wide screen, stem cell differentiation
- MeSH terms
- Gene Editing, Epigenome, Cell Differentiation, Humans, Neurons, Protein Processing, Post-Translational, CpG Islands, DNA Methylation, Gene Silencing, Histone Code, Epigenesis, Genetic, Induced Pluripotent Stem Cells, CRISPR-Cas Systems, Cellular Reprogramming
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-27.
Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-27.
Cited sources are checked by gpt-6-sol.
Statistics and arithmetic are recomputed, not judged.
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