Target residence of Cas9-sgRNA influences DNA double-strand break repair pathway choices in CRISPR/Cas9 genome editing

Abstract

Background: Due to post-cleavage residence of the Cas9-sgRNA complex at its target, Cas9-induced DNA double-strand breaks (DSBs) have to be exposed to engage DSB repair pathways. Target interaction of Cas9-sgRNA determines its target binding affinity and modulates its post-cleavage target residence duration and exposure of Cas9-induced DSBs. This exposure, via different mechanisms, may initiate variable DNA damage responses, influencing DSB repair pathway choices and contributing to mutational heterogeneity in genome editing. However, this regulation of DSB repair pathway choices is poorly understood.

Results: In repair of Cas9-induced DSBs, repair pathway choices vary widely at different target sites and classical nonhomologous end joining (c-NHEJ) is not even engaged at some sites. In mouse embryonic stem cells, weakening the target interaction of Cas9-sgRNA promotes bias towards c-NHEJ and increases target dissociation and reduces target residence of Cas9-sgRNAs in vitro. As an important strategy for enhancing homology-directed repair, inactivation of c-NHEJ aggravates off-target activities of Cas9-sgRNA due to its weak interaction with off-target sites. By dislodging Cas9-sgRNA from its cleaved targets, DNA replication alters DSB end configurations and suppresses c-NHEJ in favor of other repair pathways, whereas transcription has little effect on c-NHEJ engagement. Dissociation of Cas9-sgRNA from its cleaved target by DNA replication may generate three-ended DSBs, resulting in palindromic fusion of sister chromatids, a potential source for CRISPR/Cas9-induced on-target chromosomal rearrangements.

Conclusions: Target residence of Cas9-sgRNA modulates DSB repair pathway choices likely through varying dissociation of Cas9-sgRNA from cleaved DNA, thus widening on-target and off-target mutational spectra in CRISPR/Cas9 genome editing.

Keywords: CRISPR-Cas9 genome editing; DNA double-strand break repair pathway choice; Editing heterogeneity; Off-target effect; Target residence.

Fig. 1

Involvement of c-NHEJ varies widely in repair of Cas9-induced DSBs. a Schematic of the HDR reporter with 5 sgRNAs. Repair of Cas9-induced DSBs by HDR between sister chromatids can generate GFP⁺ cells. Inhibition of c-NHEJ is expected to promote HDR. b Effects of DNA-PKcs inhibition on SpCas9-induced HDR in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP⁺ cells; Right: Relative HDR after normalizing DMSO treatment to 1.0. c Effects of DNA-PKcs, Ku80, or Xrcc4 deficiency on SpCas9-induced HDR in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP⁺ cells; Right: Relative HDR after normalizing both WT cells and Xrcc4^+/+ cells to 1.0. d Schematic of the NHEJ reporter with 6 sgRNAs and their target sites indicated. Repair of Cas9-induced DSBs by c-NHEJ or a-EJ generates accurate NHEJ (accNHEJ) products indistinguishable from undamaged targets and mutagenic NHEJ (mutNHEJ) products represented by GFP⁺ cells. Inhibition of c-NHEJ promotes a-EJ. e Effect of DNA-PKcs inhibition and Xrcc4 deletion on SpCas9-induced NHEJ in mESC transfected with individual Cas9-sgRNA. Left: Frequencies of SpCas9-induced GFP⁺ cells; Right: Relative NHEJ after normalizing both DMSO treatment and Xrcc4^+/+ cells to 1.0. f, g Cells were transfected with SpCas9-sgRNA expression plasmids and treated with DMSO or NU7441. Four different sites of the Cola1 (f) and Rosa26 (g) locus were targeted by 4 sgRNAs indicated. The efficiency of SpCas9-induced genome editing (left) was calculated as ratios of edited reads to total reads from targeted Illumina sequencing and normalized by transfection efficiency. Relative SpCas9-induced NHEJ (right) was calculated by normalizing the editing efficiency with DMSO treatment to 1.0. Each circle indicates one independent experiment, each in triplicates. Columns indicate the mean ± S.E.M of at least three independent experiments. Significance was detected by two-tailed Student’s t test and indicated by * for P<0.05, ** for P<0.01 and *** for P<0.001

Fig. 2

Cas9 recleavage increases c-NHEJ-mediated mutations. a Model for enrichment of mutNHEJ products promoted by frequent SpCas9 recleavage with increased amount of Cas9-sgRNA transfected. With sufficient amount of SpCas9-sgRNA, accNHEJ products could be recleaved until mutNHEJ products are generated, resulting in enrichment of mutNHEJ products. b,c Effect of DNA-PKcs inhibition on NHEJ induced by varying amount of SpCas9-sgRNA. NHEJ reporter mESC were transfected with varying amounts of expression plasmids for SpCas9-gEJ_W6 (b) or SpCas9-gEJ_W7 (c) as indicated and treated with DMSO or NU7441. Frequencies of SpCas9-induced GFP⁺ cells were measured by FACS at 3 days post-transfection and relative SpCas9-induced NHEJ was calculated by normalizing DMSO treatment to 1.0. d Frequencies of GFP⁺ cells (left) and relative NHEJ (right) induced by SpCas9-sgRNA at 0.001 μg each, 1/250 of the regular amount (0.25 μg each) transfected into mESC. Each circle indicates one independent experiment, each in triplicates, and the mean of at least three independent experiments is also indicated. e,f Effect of DNA-PKcs inhibition on HDR induced by varying amount of SpCas9-sgRNA. HDR reporter mESC were transfected with varying amounts of expression plasmids for SpCas9-gHR_C2 (e) or SpCas9-gHR_C4 (f) as indicated and treated with DMSO or NU7441. Frequencies of SpCas9-induced GFP⁺ cells were measured by FACS at 3 days post-transfection and relative SpCas9-induced NHEJ was calculated by normalizing DMSO treatment to 1.0. For NHEJ and HDR assays in b–f, more than 200,000 cells were usually harvested to ensure at least 100 GFP⁺ cells counted for reliable calculation. Columns indicate the mean ± S.E.M. Statistical significance was detected by two-tailed Student’s t test: *P<0.05; **P<0.01; and ***P<0.001

Fig. 3

Reduced target binding affinity of Cas9-sgRNA shifts the pathway bias towards c-NHEJ in repair of Cas9-induced DSBs. a Effects of DNA-PKcs inhibition and Xrcc4 deletion on SpCas9-induced NHEJ in mESC transfected with individual SpCas9-sgRNA and its variants as indicated. Left: Frequencies of SpCas9-induced GFP⁺ cells; Right: Fold change of NHEJ alteration induced by NU7441 or Xrcc4 deletion relative to the SpCas9-20nt control, i.e., the ratio of NHEJ change induced by NU7441 or Xrcc4 deletion for each SpCas9-sgRNA variant to that for the SpCas9-20nt control. b, c Effects of DNA-PKcs inhibition on NHEJ-mediated genome editing at endogenous loci Cola1 (b) and Rosa26 (c) in mESC transfected with individual SpCas9-sgRNA and its variants as indicated. The frequencies of Cas9-induced indels (left) were calculated as ratios of edited reads to total reads from targeted Illumina sequencing and normalized by transfection efficiency. The fold change of NHEJ alteration induced by NU7441 relative to the SpCas9-20nt control (right) was calculated as the ratio of NHEJ change induced by NU7441 for each SpCas9-sgRNA variant to that for the SpCas9-sgRNA control. d, e Effects of DNA-PKcs inhibition and DNA-PKcs, Ku80, or Xrcc4 deficiency on Cas9-induced HDR in mESC transfected with Cas9-gHR_C4 (d), Cas9-gHR_C2 (e), and its variants as indicated. Left: Frequencies of Cas9-induced GFP⁺ cells; Right: Fold change of HDR stimulation induced by NU7441 or deletion of DNA-PKcs, Ku80, or Xrcc4 relative to the SpCas9-20nt control. This fold change was calculated as the ratio of HDR stimulation induced by NU7441 or deletion of DNA-PKcs, Ku80, or Xrcc4 for each SpCas9-sgRNA variant to that for the SpCas9-20nt control. Each circle indicates one independent experiment, each in triplicates, and the mean of at least three independent experiments is also indicated. Columns indicate the mean ± S.E.M. Statistical significance was detected by two-tailed Student’s paired t test for frequencies of Cas9-induced GFP⁺ cells or Cas9-induced indels and by one-way ANOVA followed by post hoc Dunnett’s test for fold changes of NHEJ alteration or HDR stimulation: *P<0.05; **P<0.01; and ***P<0.001

Fig. 4

Mismatched or truncated sgRNAs reduce target residence of Cas9-sgRNA in vitro at the gHR_C2, gHR_C4, gEJ_C5, and gEJ_W7 sites. The schematics for the target binding, cleavage, and dissociation reaction were shown on the top. a DNA cleavage by SpCas9-20-nt perfectly matched sgRNAs and SpCas9-sgRNA variants at different time points of the reaction as indicated. Fluorescence-labeled DNA targets were 620 bp or 623 bp as shown. DNA cleavage from the reaction was detected on 2% denaturing agarose gel. b Residence of SpCas9-sgRNAs including SpCas9-20-nt control and its variants on cleaved DNA. SpCas9-sgRNAs were incubated with fluorescence-labeled target DNAs from a for 24 h. DNA bound with SpCas9-sgRNAs and unbound DNA were resolved by 4–20% native PAGE gel. c Target residence of dSpCas9-sgRNAs including dSpCas9-20nt control and its variants. dSpCas9-sgRNAs were incubated with fluorescence-labeled target DNAs from a for 1 and 24 h. DNA bound with dSpCas9-sgRNAs and unbound DNA were resolved by 4–20% native PAGE gel. d Cleavage of DNA released from the dSpCas9-sgRNA-DNA ternary complex by competing SpCas9-20nt sgRNA. The preassembled dSpCas9-20-nt sgRNA and dSpCas9-sgRNA variant complexes were incubated with fluorescence-labeled target DNAs from a for 1 h. The preassembled SpCas9-20nt perfectly matched sgRNA complex was added to compete for binding to DNA targets released from the dSpCas9-sgRNA-DNA complex and cleave DNA for 6 and 24 h. DNA cleavage from the reaction was detected on 2% denaturing agarose gel. The efficiency of target cleavage and target dissociation was calculated as the intensity ratio of cut DNA to total DNA in a and d and the intensity ratio of unbound DNA to total DNA in b and c, respectively. The values of these ratios were shown in percentages under each DNA gel

Fig. 5

DNA-PKcs inhibition and Xrcc4 deletion aggravate off-target effect in CRISPR/Cas9 genome editing. Xrcc4^+/+ HDR reporter mESC were used for transfection with SpCas9 in complex with gPnpla3 targeting Pnpla3 (a), gMertk targeting Mertk (b), gHR_C1 (c), and gHR_C2 (d) both targeting the HDR reporter. At 6 h post-transfection, cells were treated with DMSO or NU7441. At 72 h post-transfection, gDNA was isolated and the indel frequency at on-target and selected off-target sites was measured by amplicon deep sequencing and calculated as the ratio of edited reads to total reads normalized by transfection efficiency. In an independent set of experiments, isogenic Xrcc4^+/+ and Xrcc4^–/– HDR reporter mESC were transfected and the indel frequency at on-target and selected off-target sites was similarly measured. Fold change of off-target effect after treatment of NU7441 or deletion of Xrcc4 was calculated as the ratio of the indel frequency with treatment of NU7441 or in Xrcc4^–/– cells to that with DMSO or in Xrcc4^+/+ cells at each off-target site, respectively. Each circle indicates one independent experiment, and the mean of these independent experiments is also indicated. Error bars indicate S.E.M. Statistical analysis was performed by two-tailed Student’s paired t test for frequencies of Cas9-induced indels and by one-way ANOVA followed by post hoc Dunnett’s test for fold changes of off-target effect between NU7441 and DMSO, and between Xrcc4^+/+ and Xrcc4^–/–. *P<0.05 and **P<0.01

Fig. 6

Transcription has no effect on c-NHEJ in repair of Cas9-induced DSBs. a dSpCas9-mediated transcriptional silencing in mESC containing pPGK-GFP expression cassette. The mean fluorescence intensity of GFP indicates relative transcription. b Involvement of c-NHEJ in SpCas9-induced DSBs in mESC containing pPGK-GFP expression cassette. Cells were transfected with a low amount of individual SpCas9-sgRNA expression plasmids (0.001 μg SpCas9, 0.001 μg sgRNA, 1/500 of total DNA each) as shown and treated with NU7441 at 6 h post-transfection. Relative NHEJ (n=3) was calculated by normalizing DMSO treatment to 1.0. c Analysis of strand bias in transcriptional silencing (top) and c-NHEJ involvement (bottom) between transcription template strand and transcription non-template strand targeted by dSpCas9-sgRNA or SpCas9-sgRNA. Transcriptional silencing and c-NHEJ involvement were defined as the percentage of GFP fluorescence intensity reduced by dSpCas9-sgRNA and the percentage of SpCas9-induced NHEJ stimulated by NU7441, respectively. d Correlation between dSpCas9-mediated transcriptional silencing and c-NHEJ involvement in repair of SpCas9-induced DSBs. Each circle indicates the level of dSpCas9-mediated transcriptional silencing and stimulation of SpCas9-induced NHEJ by NU7441 at the same target. Two sgRNAs gG_W5 and gG_C4 are indicated by arrows for their strong effect on transcriptional silencing. e dSaCas9-mediated transcriptional silencing in mESC containing pPGK-GFP expression cassette. The mean fluorescence intensity of GFP indicates relative transcription. Transcription blockage by dSaCas9-gSaG_W1 and dSaCas9-gSaG_W2 induced significant transcription silencing. Positions of the targets by dSaCas9-sgRNAs are indicated in the reporter. f Little effect of transcription blockage by dSaCas9-gSaG_W1 and dSaCas9-gSaG_W2 on DNA-PKcs involvement in SpCas9-mediated GFP gene editing. GFP⁺ cells were co-transfected with SpCas9-sgRNA (SpCas9 and sgRNA at 0.125 μg and 0.0005 μg respectively) and dSaCas9-sgRNA (0.125 μg each), and frequencies of SpCas9-induced GFP^– cells measured by FACS at 4 days post-transfection. Each circle indicates one independent experiment, and the mean of these independent experiments is also indicated. Error bars indicate S.E.M. Two-tailed Student’s paired or unpaired t test is indicated by * for P<0.05, ** for P<0.01, *** for P<0.001, and n.s. for not significant. Correlation between transcription silencing and the NHEJ increase was determined by linear regression analysis

Fig. 7

Replication adjacent to targets of Cas9-sgRNA suppresses c-NHEJ in repair of Cas9-induced DSBs. a Impact of local replication on DNA-PKcs involvement in NHEJ. SV40 LT can bind to the SV40 origin in a Firefly luciferase-based NHEJ reporter (Luc: Firefly luciferase; SV40 ori: SV40 DNA replication origin) to initiate replication during DNA cleavage by I-SceI or SpCas9-gEJ_W10 in 293 cells. NHEJ is represented as relative luciferase activity (i.e., ratio of Firefly luciferase activity to Renilla luciferase activity). Percentage of NHEJ reduction is indicated above each column. b Impact of local replication on DNA-PKcs involvement in HDR induced by I-SceI (left) or SpCas9-gHR_C2 (right) in HDR reporter U2OS cells. SV40 LT expressed can bind to the SV40 origin in the HDR reporter to initiate replication. SV40 LT was titrated as indicated. The fold of the increase is shown above each column. c Analysis schematic for SpCas9-induced HDR and NHEJ at the same site of the HDR reporter. HDR bias: HDR reads/ (HDR and NHEJ reads). d Effect of Cas9-sgRNA target binding on HDR bias in repair of Cas9-induced DSBs in HDR reporter mESC (left) and U2OS cells (right). e Detection schematic for three ends generated by a collision between a DNA replication fork and Cas9-sgRNA at cleaved target. Three primers with different distance to the end, TF1, TF2, and TF3, were screened in pairs for PCR as indicated. f PCR detection of palindromic sister chromatid ligation in HDR reporter mESC and U2OS cells. Expression of SpCas9-gHR_C4, empty vector control, and SV40 LT is indicated. PCR was performed with the primer pair of TF1 and TF2 on gDNA. g Repair junction of sister chromatid ligation by subcloning of PCR products and Sanger sequencing. Only two types of products (#1 and #2) were detected with the size and position of deletion (del) and insertion (ins) as indicated. *T: insertion of a thymidine nucleotide. h Impact of Cas9-sgRNA target residence on local repair pathway choice. Each circle indicates one independent experiment, each in triplicates, and the mean of at least three independent experiments is also indicated. Columns indicate the mean ± S.E.M. Statistical significance was detected by two-tailed Student’s t test: *P<0.05; **P<0.01; and ***P<0.001