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

To directly analyze the extent of c-NHEJ involvement in repair of Cas9-induced DSBs at different target sites, we used Cas9-sgRNA to induce site-specific DSBs in an NHEJ reporter integrated in the genome of mESC as done before [28] and analyzed the effect of c-NHEJ inactivation on the frequencies of Cas9-induced insertion or deletion mutations (indels) (Fig. 1d and Additional file 1: Fig. S2a). In this NHEJ reporter, no wild-type GFP is translated due to an upstream, out-of-frame translation start site (Koz-ATG), which is flanked by two I-SceI sites sequentially positioned [29]. When a DSB is induced by Cas9-sgRNA at a site between "Koz-ATG" and the ATG-GFP coding region, repair by either c-NHEJ or a-EJ can generate indels at the repair junction. In general, because of a 34-bp interval between "Koz-ATG" and ATG for GFP, indels with net addition of "3n+2" bp or net loss of "3n-1" bp can change the 34-bp frame-shift to in-frame, leading to production of GFP⁺ cells. The frequency of Cas9-induced GFP⁺ cells thus represents the relative efficiency of Cas9-induced indels [28] (Fig. 1d and Additional file 1: Fig. S2a). As c-NHEJ and a-EJ generate different proportions of accurate NHEJ (accNHEJ) products and indel-based mutagenic NHEJ (mutNHEJ) products [4], inactivation of c-NHEJ would channel more Cas9-induced DSBs towards error-prone a-EJ in addition to HDR, increasing the frequencies of mutNHEJ. We found that neither DNA-PKcs inhibition by NU7441 nor Xrcc4 deletion changed the frequencies of mutNHEJ represented by Cas9-induced GFP⁺ cells at the two sites targeted by the sgRNA gEJ_W3 or gEJ_W7, suggesting little involvement of c-NHEJ at these two sites (Fig. 1e). However, inactivation of c-NHEJ inhibited the level of Cas9-induced GFP⁺ cells at the four sites targeted by gEJ_C5, gEJ_W4, gEJ_W5, and gEJ_W6 to different extents, varying from 16.6 to 69.2% (Fig. 1e). Consistently, junction analysis by targeted PCR amplicon deep sequencing revealed that NU7441 had little or modest effect on the editing efficiency, the length distribution of deletions, and the MH usage at the gEJw7 site, but significantly altered the editing efficiency, the length distribution of deletions, and the MH usage at the gEJc5 site (Additional file 1: Fig. S2b-d). As a dominant type of Cas9-induced indels [6, 18, 30 –32], 1-bp templated insertions (TIs), which do not generate GFP⁺ cells in NHEJ reporter cells, occurred much more frequently at the gEJw7 site than at the gEJc5 site (Additional file 1: Fig. S2e). Together, these results indicate that the participation of c-NHEJ varies in repair of Cas9-induced DSBs at different targets in mESC.

Using targeted PCR amplicon deep sequencing, we also measured the frequencies of Cas9-induced indels at two natural genome loci Cola1 and Rosa26 in mESC. We found that NU7441 reduced the editing efficiency at the sites targeted by Cola1 gC2 and Rosa26 gR3, stimulated by more than 2-fold at the sites by Cola1 gC3, and had minimal effect at the rest of the sites including gC1 and gC4 for Cola1 and gR1, gR2, and gR4 for Rosa26 (Fig. 1f, g). Generally, DNA-PKcs inhibition had little or modest effect on the length distribution of deletions and the MH usage at the target sites where c-NHEJ involvement is limited, but increased deletion length and the MH usage at the target sites where c-NHEJ is engaged (Additional file 1: Fig. S3a-c). Together with varying stimulation of Cas9-induced HDR at different targets by inactivation of c-NHEJ, these results suggested variable involvement of c-NHEJ in CRISPR/Cas9 genome editing at different sites or even no involvement of c-NHEJ at some sites in mESC.

Like I-SceI, CRISPR nucleases generate DSBs with directly ligatable ends. Previous studies have demonstrated that c-NHEJ is intrinsically accurate for these ends [6, 7, 29]. In each round of repair during CRISPR/Cas9 genome editing, about a half of NHEJ products are accurate in repair of Cas9-induced DSBs and the remaining half generate indels [6, 8, 9]. Thus, inactivation of c-NHEJ would increase the use of a-EJ in each round of CRISPR/Cas9 genome editing. Since a-EJ is more error prone, inactivation of c-NHEJ would elevate Cas9-induced indels. It is unexpected that the frequency of Cas9-induced indels was instead inhibited at many Cas9-sgRNA target sites by inactivation of c-NHEJ (Fig. 1e–g). To determine whether this was unique to repair of Cas9-induced DSBs, we used the same NHEJ reporter cells but with the first I-SceI site being deleted to ensure that I-SceI induces single cleavage as Cas9 does and compared the effect of c-NHEJ inactivation on the frequency of Cas9- and I-SceI-induced indels represented by GFP⁺ cells (Additional file 1: Fig. S4a). In consistent with previous findings that inactivation of c-NHEJ stimulates production of I-SceI-induced GFP⁺ cells [28, 33], inhibition of c-NHEJ with NU7441 increases I-SceI-induced GFP⁺ cells by more than 2-fold (Additional file 1: Fig. S4b,c). Given the fact that inactivation of c-NHEJ suppresses Cas9-induced indels at many Cas9-sgRNA target sites, this appears to suggest a difference between Cas9- and I-SceI-NHEJ.

We then reassessed the c-NHEJ engagement at the 6 Cas9-sgRNA target sites when Cas9 recleavage of the regenerated target is prevented by lowering the transfection amount of Cas9-sgRNA. At the two sites targeted by gEJ_W4 and gEJ_W6 with the transfection amount of Cas9-sgRNA at 0.001 μg, Cas9-induced indels were also reduced as expected (Fig. 2d). DNA-PKcs inhibition and Xrcc4 deletion did not suppress production of Cas9-induced GFP⁺ cells any more or even reversed to stimulation at the gEJ_W4 and gEJ_W6 targets but remained to exert no effect on the level of Cas9-induced GFP⁺ cells at the gEJ_W3 or gEJ_W7 site (Fig. 2d). In fact, Xrcc4 deletion elevated the frequency of Cas9-induced GFP⁺ cells by 59.6 ± 14.2% (P<0.05) at the gEJ_W4 target and 81.5 ± 24.5% (P<0.05) at the gEJ_W6 target with 0.001 μg of Cas9-sgRNA (Fig. 2d), a reverse from reduction of Cas9-induced GFP⁺ cells by 58.1 ±3% and 60.4 ± 2.4% respectively at these two targets with 0.25 μg of Cas9-sgRNA (Fig. 1e). These results again indicate that limiting Cas9 recleavage could elicit the stimulatory effect of c-NHEJ inactivation on Cas9-induced indels in mESC.

Differently, at the gEJ_C5 or gEJ_W5 target, with the transfection amount of Cas9-sgRNA at 0.001 μg, DNA-PKcs inhibition and Xrcc4 deletion still inhibited the generation of Cas9-induced GFP⁺ cells; but this inhibition was reduced (Fig. 2d). At the gEJ_C5 target, Xrcc4 deletion reduced Cas9-induced GFP⁺ cells by 37.0 ±3.2% (P<0.001) with 0.001 μg of Cas9-sgRNA, a smaller reduction than 55.8 ± 2.6% (P<0.001) with 0.25 μg of Cas9-sgRNA (Figs. 2d vs. 1e). At the gEJ_W5 target, this reduction of GFP⁺ cells by Xrcc4 deletion is 57.6 ± 7.5% (P<0.001) with 0.001 μg of Cas9-sgRNA but 69.2 ±1.5% (P<0.01) with 0.25 μg of Cas9-sgRNA (Figs. 2d vs. 1e). This suggests that Cas9 recleavage could still abrogate the stimulatory effect of c-NHEJ inactivation on Cas9-induced indels at the gEJ_C5 or gEJ_W5 target sites where limiting Cas9 recleavage does not fully abolish the suppression of Cas9-induced indels by c-NHEJ inactivation. Similar to the gEJ_W7 target, no effect by c-NHEJ inactivation was detected at the gEJ_W3 target with neither 0.001 nor 0.25 μg of Cas9-sgRNA (Figs. 2d and 1e), suggesting no engagement of c-NHEJ at these two sites. Taken together, these results not only indicate that target recleavage by Cas9 amplifies the mutagenicity of c-NHEJ in CRISPR/Cas9 genome editing in mESC, but also confirm that the involvement of c-NHEJ varies significantly at different targets in repair of Cas9-induced DSBs after target recleavage by Cas9 is partially or fully prevented. Of note, DNA-PKcs inhibition by NU7441 elicited weaker manifestation of c-NHEJ engagement than deletion of Xrcc4. This is possible due to different functions of these two factors in NHEJ.

Since minimizing Cas9 recleavage helps maintain a higher proportion of accurate NHEJ products among NHEJ events if c-NHEJ is engaged in repair of Cas9-induced DSBs, inactivation of c-NHEJ would stimulate HDR more strongly. Indeed, at the gHR_C2 site, the HDR stimulation by NU7441 was enhanced after the transfection amount of Cas9-sgRNA was reduced (Fig. 2e), confirming c-NHEJ engagement in repair of Cas9-induced DSBs at this site. In contrast, at the gHR_C4 site, HDR was not stimulated by NU7441 regardless of the transfection amount of Cas9-sgRNA (Fig. 2f), again indicating that c-NHEJ is little involved at this site.

Using endogenous genomic loci, we also found that the editing efficiency with the mismatch variants of Cola1 gC4 (i.e., C1T and G16C) and Rosa26 gR4 (i.e., A1C and A16T) was reduced due to weaker target interaction (Fig. 3b, c and Additional file 1: Fig. S5b). Consistently, DNA-PKcs inhibition by NU7441 had minimal effect on Cas9-induced indels at the sites targeted by Cola1 gC4 and Rosa26 gR4 (Fig. 1f, g), but stimulated Cas9-induced indels with the gC4 variant G16C and the gR4 variants A1C and A16T (Fig. 3b, c and Additional file 1: Fig. S5b). This again indicates that reducing Cas9-sgRNA target interaction promotes c-NHEJ. Taken together, these results suggest that weakened target interaction of Cas9-sgRNA increase bias towards c-NHEJ in repair of Cas9-induced DSBs in mESC.

Consistently with previous studies [23 –25], inactivation of c-NHEJ stimulates HDR induced by CRISPR nucleases as well as I-SceI (Fig. 1b, c and Additional file 1: Fig. S1d). We expected that this stimulatory effect would be further enhanced if HDR were induced by Cas9-sgRNA variants with reduced target interaction, because reducing Cas9-sgRNA target interaction promotes c-NHEJ. We thus compared HDR induced by mutated Cas9-sgRNA between cells proficient and deficient in c-NHEJ. Due to reduced efficiency of DNA cutting, Cas9-induced HDR was generally less efficient with mismatched or truncated sgRNA variants (i.e., G1C, G2C, and 17nt for gHR_C4, and A1T, C2A, and 17nt for gHR_C2) and SpCas9 variants eSpCas9, SpCas9-HF1, and xCas9 (i.e., xCas9-3.7), except eSpCas9-gHR_C4, SpCas9-gHR_C2 17nt, and xCas9-gHR_C2 20nt (Fig. 3d, e and Additional file 1: Fig. S5c).

At the site targeted by gHR_C4, as in Fig. 1c, Cas9-induced HDR was not affected by DNA-PKcs inhibition, DNA-PKcs deletion, or Ku80 deletion, but modestly stimulated by deletion of Xrcc4, Cas9-induced HDR with the sgRNA variants G2C and 17nt was elevated by NU7441 (Fig. 3d). Similarly, deletion of DNA-PKcs or Ku80 elicited stimulatory effect on Cas9-induced HDR with gHR_C4 G1C and 17nt, as well as with SpCas9-HF1 (Fig. 3d). In addition, Xrcc4 deletion stimulated Cas9-induced HDR with the gHR_C4 variants (i.e., G1C, G2C, and 17nt) and the SpCas9 variants SpCas9-HF1 and xCas9 by up to 4.3-fold (Fig. 3d). At the site targeted by gHR_C2, where Cas9-induced HDR was increased by DNA-PKcs inhibition or deletion of DNA-PKcs, Ku80, or Xrcc4 as in Fig. 1c, stimulation of Cas9-induced HDR by NU7441 was further enhanced with the SpCas9 variants such as eSpCas9 and SpCas9-HF1 (Fig. 3e). This HDR stimulation for the SpCas9 variants increased by 1.2- to 2.2-fold as compared to the SpCas9 control (Fig. 3e). Deletion of DNA-PKcs, Ku80, or Xrcc4 caused more stimulation of Cas9-induced HDR for SpCas9-gHR_C2 C2A, eSpCas9-20nt, and SpCas9-HF1-20nt as this HDR stimulation were enhanced by up to 4.9-fold (Fig. 3e).

However, neither DNA-PKcs inhibition nor genetic inactivation of c-NHEJ by deletion of DNA-PKcs, Ku80, or Xrcc4 stimulated more HDR induced by eSpCas9-gHR_C4 20nt, SpCas9-gHR_C2 A1T, SpCas9-gHR_C2 17nt, or xCas9-gHR_C2 20nt than that induced by their respective SpCas9-20nt controls (Fig. 3d, e). It appeared that HDR induced by these Cas9-sgRNA variants is as efficient as that by their SpCas9-20nt controls at their target sites (Fig. 3d, e). It is possible that little is changed in the strength of target interaction or the efficiency of target cleavage between the SpCas9-20nt control and Cas9-sgRNA variants at these sites despite modification of SpCas9 or sgRNA. Taken together, these results above confirm that reducing target interaction of Cas9-sgRNA promotes c-NHEJ in mESC, providing the basis for the enhanced stimulatory effect of c-NHEJ inactivation on Cas9-induced HDR.

For fully matched 20-nt sgRNAs, dSpCas9, sgRNA, and its target DNA at the molar ratio of 5:5:1 assembled nearly all target DNA into dSpCas9-sgRNA-DNA ternary complex in vitro at 1 h and 24 h of the reaction, leaving little unbound DNA (Fig. 4c). By comparison, a significant level of target DNA remained unbound by dSpCas9-sgRNA variants at 1 h and 24 h of the in vitro assembly (Fig. 4c). This again suggests that reducing target interaction could either lessen initial target binding or facilitate target dissociation of SpCas9-sgRNA. To determine whether uncleaved target DNA could be released from the dSpCas9-sgRNA-DNA ternary complex, we added SpCas9-20nt into the mix of the in vitro assembly reaction immediately after the dSpCas9-sgRNA-DNA complex was assembled for 1 h, allowing the competing SpCas9-20nt to cleave the initially unbound DNA or the DNA newly released from the dSpCas9-sgRNA-DNA complex for 6 h and 24 h (Fig. 4d). Little or modestly some target DNA from the dSpCas9-20nt-DNA complex was cleaved by SpCas9 at all 4 target sites, i.e., the gHRc2, gHRc4, gEJc5, and gEJw7 sites, indicating target protection by persistent target residence of dSpCas9 complexed with fully matched 20-nt sgRNA (Fig. 4d). In contrast, a portion of target DNA from the in vitro dSpCas9-sgRNA-DNA ternary complex assembly containing mismatched or truncated sgRNAs was cleaved by SpCas9 at 6 h of the cleavage reaction (Fig. 4d). The level of cleavage by SpCas9 increased further at 24 h for all mismatched or truncated sgRNAs (Fig. 4d), indicating continuous dissociation of target DNA from the dSpCas9-sgRNA-DNA complex with mismatched or truncated sgRNAs. These results again suggest that reducing target interaction of Cas9-sgRNA promote target dissociation and shorten target residence of SpCas9-sgRNA.

Chemical inhibition and genetic inactivation of c-NHEJ are often used to increase the efficiency of Cas9-induced HDR-mediated gene knock-in or replacement [40 –45]. Given that DNA-PKcs inhibition by NU7441 stimulated Cas9-induced HDR in the HDR reporter at the targets by gHR_C1 and gHR_C2 (Fig. 1b), we also performed off-target analysis for 6 potential off-target sites for Cas9-gHR_C1 and Cas9-gHR_C2 in mESC, respectively. After NU7441 treatment, the frequencies of on-target indels induced by Cas9-gHR_C1 and Cas9-gHR_C2 were slightly lowered by 20–40%, again indicating significant on-target DNA recleavage. Unlike on-target editing, the frequencies of Cas9-induced indels at the 6 off-target sites were not reduced by NU7441. Instead, these frequencies were stimulated by DNA-PKcs inactivation or Xrcc4 deletion (Fig. 5c,d), and the fold change of off-target effect was elevated up to 2.5 (Fig. 5c,d). This again suggests that both chemical inhibition and genetic inactivation of c-NHEJ exacerbate off-target effects in CRISPR/Cas9 genome editing in mESC.

To further determine whether lack of c-NHEJ engagement in repair of Cas9-induced DSBs at some sites is caused by transcription-mediated Cas9-sgRNA target dissociation generating ends unsuitable for c-NHEJ, we analyzed the effect of local transcription blockage on the state of c-NHEJ engagement at a given site where c-NHEJ is little involved. We thus used catalytically dead Staphylococcus aureus Cas9 (dSaCas9)-sgRNA to block the translocating RNA polymerase (RNAP), preventing downstream dissociation of SpCas9-sgRNA from its cleaved target (Fig. 6e). Among 6 sgRNAs tested for transcriptional blockage, only gSaG_W1 and gSaG_W2, in complex with dSaCas9, efficiently reduced gene expression by 26.7±4.5% (P<0.05) and 47.4± 7.3% (P<0.01) respectively, indicating a strong capability of blocking RNAP (Fig. 6e). As in Fig. 6b, the frequency of GFP^– cells induced by SpCas9-gG_C4 and SpCas9-gG_W5 at a transfection amount of 0.125 or 0.0005 μg for each plasmid was not altered by DNA-PKcs inhibition with NU7441, indicating little c-NHEJ involvement in repair of Cas9-induced DSBs at these two sites (Fig. 6f). This lack of c-NHEJ engagement was little changed by co-transfection with either dSaCas9-gSaG_W1 or dSaCas9-gSaG_W2 that could block local transcription (Fig. 6f). This indicates that transcription blockage upstream by dSaCas9-sgRNA (e.g., dSaCas9-gSaG_W1 and dSaCas9-gSaG_W2) would not promote c-NHEJ engagement in repair of SpCas9-induced DSBs at sites where c-NHEJ is little engaged and further excludes the possibility that transcription-mediated Cas9-sgRNA target dissociation prevent c-NHEJ engagement in repair of Cas9-induced DSBs in mESC.

We also wondered whether a collision with local DNA replication would favor HDR over c-NHEJ in repair of Cas9-induced DSBs by blocking c-NHEJ engagement, thus removing the stimulatory effect of DNA-PKcs inhibition on Cas9-induced HDR. Using U2OS cells containing an integrated single-copy HDR reporter (Fig. 7b), in which an SV40 origin is located between TrGFP and I-SceI-GFP, we analyzed the effect of DNA-PKcs inhibition by NU7441 on HDR induced by I-SceI and SpCas9. In consistent with the results from mESC (Fig. 1b), NU7441 stimulated HDR induced by SpCas9 in complex with gHR_C1, gHR_C2, gHR_C3, gHR_C4, and gHR_C5 to different degrees, as well as by I-SceI (Fig. 7b and Additional file 1: Fig. S7a), indicating variable but detectable engagement of the competing c-NHEJ pathway in repair of these I-SceI- or Cas9-induced DSBs. After expression of SV40 LT, HDR induced by I-SceI, Cas9-gHR_C2, Cas9-gHR_C3, and Cas9-gHR_C4 were repressed in a gradual and dose-dependent manner (Fig. 7b and Additional file 1: Fig. S7b,c). NU7441 stimulated I-SceI- or Cas9-induced HDR, and the expression of SV40 LT attenuated this stimulation of I-SceI-induced HDR from 4.9-fold to 2.5-fold or even abolished the NU7441-induced stimulation of Cas9-induced HDR at a transfection amount of 0.032 μg (1/25 of total DNA transfected) for Cas9-gHR_C4 and 0.16 μg (1/5 of total DNA transfected) for Cas9-gHR_C2 (Fig. 7b and Additional file 1: Fig. S7b,c). This suggests that local DNA replication driven by SV40 LT could collide with both I-SceI and SpCas9-sgRNA after DNA cleavage to dislodge I-SceI and Cas9-sgRNA from its cleaved target and restrict the engagement of c-NHEJ in repair of exposed DSBs.

By restricting c-NHEJ due to a collision with replication fork, DSB repair pathway choice would be biased towards HDR. To test this possibility, we used the HDR reporter to measure the bias between HDR and NHEJ in repair of the same DSB induced by SpCas9-sgRNA that was tightly bound with its target and by SpCas9-sgRNA variants with weakened target interaction. In the HDR reporter, repair of the same Cas9-induced DSBs around the I-SceI site of I-SceI-GFP by HDR generates the "WT GFP," whereas NHEJ generates "mutant GFP" due to disruption of the I-SceI site (Fig. 7c). We can separate these two repair outcomes in mESC by nested PCR and evaluate the HDR bias (i.e., the ratio of HDR to total edited) by deep sequence analysis. After HDR and NHEJ induced by SpCas9-gHR_C4 in mESC, we found the HDR bias was nearly 3-fold lower with gHR_C4 variants (G1C and 17nt) than with gHR_C4 (Fig. 7d), indicating a reduced HDR preference when the interaction of SpCas9-sgRNA to its target is weakened. At the site targeted by gHR_C2, where the HDR stimulation by DNA-PKcs inhibition was fully abolished in U2OS cells by the expression of SV40 LT at a transfection amount of 0.16 μg (Fig. 7b), SV40 LT expression at the same transfection amount increased the HDR bias by nearly 2-fold (Fig. 7d), indicating a shift of the repair pathway from NHEJ to HDR. Therefore, for Cas9-sgRNA target sites where c-NHEJ is disfavored in repair of Cas9-induced DSBs, it is likely that Cas9-sgRNA at these sites may have a higher probability for collision with local DNA replication after DNA cleavage due to persistent target interaction. Cas9-induced replication-coupled DSBs are subsequently generated with particular end configurations in S phase and favor HDR over c-NHEJ for their repair.

While spontaneous dissociation of Cas9-sgRNA from cleaved DNA results in a conventional two-ended DSB, DNA replication that releases Cas9-sgRNA from its cleaved target may generate a three-ended DSB, with the leading strand likely forming a blunt end on one sister chromatid and the lagging strand a 3′-overhanging end with long ssDNA on the other sister chromatid (Fig. 7e). These two ends each can rejoin with the other blunt end of the DSB, or have a potential to directly ligate with each other, the latter generating a palindromic chromosome from sister chromatid fusion (SCF) and potentially promoting chromatid breakage-fusion-bridge (BFB) cycles [46 –49] (Fig. 7e). Because neither DNA-PKcs nor Ku80 is engaged at Cas9-induced DSBs at the gHR_C4 target site for repair in the HDR reporter in mESC (Fig. 1b, c), it is likely that Cas9-gHR_C4 at this site may collide with a replication fork after DNA cleavage, generating a three-ended DSB and allowing subsequent fusion of two sister chromatids and production of a palindromic chromosome. Because the product contains palindromic DNA sequence surrounding the junctions, a single primer could in theory be annealed to both the leading strand template and the newly synthesized lagging strand in the repair product for PCR amplification. However, no PCR products were detected from repair of Cas9-induced DSBs at the gHR_C4 target site in the HDR reporter in mESC and U2OS cells with a single primer, e.g., TF1, TF2, or TF3 (data not shown), likely due to replication slippage of Taq DNA polymerase over a hairpin structure formed by palindromic DNA sequences in PCR amplification [50, 51]. We thus paired a distal primer to the break (TF2 or TF3) with the most proximal primer TF1 to minimize the length of palindromic DNA sequence in PCR amplification of repair products induced by SpCas9-gHR_C4 in the HDR reporter and detected PCR bands over 250 bp in mESC (Fig. 7f and Additional file 1: Fig. S8a). In U2OS cells, these PCR bands were detected only after expression of SV40 LT, suggesting replication-coupled generation of three-ended DSBs and fusion of newly duplicated sister chromatids at the SpCas9-gHR_C4 cleavage site (Fig. 7f and Additional file 1: Fig. S8a). This is consistent with the observation that DNA-PKcs inhibition stimulates HDR induced by Cas9-gHR_C4 in U2OS cells, but neither in mESC nor in U2OS cells highly expressing SV40 LT.

To further confirm that the PCR bands for these repair products were indeed fusions of sister chromatids via end ligation of Cas9-induced DSBs, we first cloned PCR products into a plasmid for Sanger sequencing. Among 40 clones for PCR bands with TF1 and TF2, 17 were from mESC and the rest from U2OS cells. Among 31 clones for PCR bands with TF1 and TF3, 29 were from mESC and the rest from U2OS cells. Sanger sequencing revealed only two sequence variations in each PCR band: DL251R6 and DL268R1 for the PCR band with TF1 and TF2 and DL231R5 and DL386R45 for the PCR band with TF1 and TF3 (Fig. 7g and Additional file 1: Fig. S8b). They all contained some GFP sequences inverted around the break site but no palindromic GFP sequences, indicating that SCF occur but palindromic sequences may be lost during repair or may not be amplified by PCR [50 –54] (Additional files 2, 3 and 4: Table S1-S3). The deletion length in each sequence was distinctly asymmetric surrounding the break point, long at 231bp, 251bp, 268bp, or 386bp at one direction and short at 1bp, 5bp, 6bp, or 45bp at the other direction (Fig. 7g and Additional file 1: Fig. S8b). Lack of palindromic sequences in these PCR products is consistent with previous studies [47, 52 –54]. It is likely that the collision between DNA replication and Cas9-sgRNA could generate long ssDNA at the lagging strand end and little or no ssDNA overhang at the leading strand end. Long ssDNA could be easily degraded, generating long deletion and loss of palindromic sequences. It is also possible that one half of palindromic sequences is lost in the first-round PCR due to replication slippage of Taq DNA polymerase over a hairpin structure formed by palindromic sequences [50, 51]. PCR targeted amplicon sequencing also confirmed inverted GFP sequences with no palindromic fragments around Cas9-induced DSBs, but with more junction sequence variations (Additional file 1: Fig. S9a,b). Taken together, these results suggest that three-ended DSBs could be generated from release of Cas9-sgRNA at some cleaved targets upon encountering local DNA replication, resulting in inverted duplication via end-joining of sister chromatids.

The expression plasmids for truncated and mismatched sgRNAs were constructed as described [37], and the expression plasmids for SpCas9, SpCas9 variants eSpCas9, SpCas9-HF1 and xCas9-3.7, and d SpCas9 were constructed previously [38, 39, 70]. The sgRNA target sequences and respective mutations for SpCas9 and SaCas9 are listed in Additional file 5: Table S4. The HDR reporter plasmid was previously constructed [26, 71]. To generate the reporter plasmid GFP-P2A-FLuc for replication fork-SpCas9 collision assays, the P2A-Firefly luciferase (FLuc) gene was fused to C-terminal of GFP in the sGEJ reporter previously established [29]. Due to an SV40 replication origin originally present in the sGEJ reporter, DNA replication can be induced by expression of SV40 LT in the GFP-P2A-FLuc collision reporter.

HEK293 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM L-glutamine. mESC were cultured as described before [71]. Isogenic Xrcc4^+/+ and Xrcc4^–/– mESC containing the NHEJ reporter and the HDR reporter were established previously [27, 29]. Using a different line of HDR reporter mESC [26], DNA-PKcs^–/– and Ku80^–/– HDR reporter mESC along with isogenic wild-type clones were generated by the paired Cas9-sgRNA method as previously described [6]. Briefly, 2×10⁵ mESC were transfected with the expression plasmids for paired sgRNAs and Cas9 in a 24-well plate and were seeded on mouse embryonic fibroblast (MEF) feeder cells for single clones without any antibiotic selection. Knock-out clones were verified by PCR along with Sanger sequencing and Western blot. The HDR reporter U2OS cells were generated as previously described [72]. To generate the GFP⁺ cell lines for GFP KO experiments, mESC harboring the NHEJ reporter were transfected with expression plasmids for SpCas9-gI-SceI site and GFP⁺ cells were cloned, expanded, and determined by fluorescence-activated cell sorting (FACS) using the Beckman Coulter CytoFLEX flow cytometer.

Transfection of mESC was done with Lipofectamine 2000 (Invitrogen) in 24-well plates as previously described [28, 71]. Total 2×10⁵ mESC harboring the HDR/NHEJ reporter in 200 μL culture medium were transfected with the expression plasmids for Cas9 (0.25 μg) and sgRNA (0.25 μg) or for the pcDNA3β control (0.5 μg). Transfection efficiencies were measured by parallel transfection of 0.05 μg pcDNA3β-GFP expression vector together with 0.45 μg the empty vector pcDNA3β. We excluded experiments in poor transfection efficiencies that were less than 50%. The HDR/NHEJ frequencies were calculated after being corrected by background readings (the pcDNA3β control) and normalized by the transfection efficiencies (the pcDNA3β-GFP vector). Cells were analyzed for the frequencies of induced GFP⁺ cells at least 3 days post-transfection but the transfection efficiencies were determined at no more than 3 days post-transfection. For U2OS or HEK293 cells transfection, 1.0×10⁵ cells were seeded on a 24-well plate and grown to 80–95% confluence. 0.8 μg total DNA were transfected by Lipofectamine 2000. Cells harboring the NHEJ or HDR reporter were transfected with pcDNA3β-I-SceI or the expression plasmids for SpCas9-sgRNA or SaCas9-sgRNA as previously described [28, 71].

In dSaCas9-sgRNA transcription blockage experiments, GFP⁺ mESC were transfected with the expression plasmids for SpCas9-sgRNA, together with the expression plasmids for dSaCas9-sgRNA. In replication fork-dSpCas9 collision experiments, cells were transfected with the expression plasmids for I-SceI or SpCas9-sgRNA and the SV40 LT, together with the GFP-P2A-FLuc reporter plasmid as needed. If necessary, cells were treated with DNA-PKcs inhibitor NU7441 (TopScience Cat# T6276) at 6 h post-transfection. NU7441 was replaced with a fresh addition of the drug the next day. GFP⁺ and GFP^– cells were determined by FACS at 72 h and 96 h respectively post-transfection. The frequencies of NHEJ, HDR, and genome editing were calculated after being corrected with background readings and normalized with transfection efficiencies as described before [28].

To evaluate the effect of Cas9 dosage on NHEJ, NHEJ reporter cells were transfected with a varying amount of Cas9-sgRNA each at 0.25 μg, 0.1 μg, 0.01 μg, 0.001 μg, and 0.0001 μg. Cells transfected were treated with 2.5 μM NU7441 and analyzed by FACS 3 days post-transfection.

The in vitro DNA cleavage and EMSA were performed as described previously [13, 14, 55]. SpCas9 nuclease (Z03386, 0.2 μg/μL) was purchased from GenScript Biotech. The dSpCas9 nuclease (PC1351, 0.5 μg/μL) was purchased from Inovogen Biotech. All sgRNAs used for SpCas9 and dSpCas9 were synthesized by GenScript Biotech and transported in powder packages. The sgRNAs were dissolved in RNA-free water and diluted to 1 μM before use. The primers labeled with either 5′-DyLight-680 or 5′-DyLight-800 were purchased from Takara BioMed (Additional file 6: Table S5). PCR was performed to generate 600–700 bp fluorescence-labeled DNA fragments. For a standard DNA cleavage reaction, 0.5 pmol SpCas9 was mixed with 0.5 pmol sgRNA in Cas9 nuclease reaction buffer (GenScript) and was incubated for 10 min at 37 °C. Then 0.1 pmol target DNA fluorescently labeled was added subsequently, making a total volume of 10 μL with RNA-free water. The reaction was performed at 37 °C and quenched by the addition of 2 μL of denatured loading dye. The cleaved DNA was resolved by 2% agarose gel electrophoresis for fluorescence-imaging analysis.

Similarly, in the EMSA assays, the binding reaction with target DNA was performed with 0.1 pmol target DNA after the dSpCas9-sgRNA complex was assembled with 0.5 pmol dSpCas9 and sgRNA each for 1 h or 24 h. The samples were resolved on 4–20% SurePAGE non-denatured gel (GenScript) in 0.5×TBE buffer at 200 V for 150 min in 4 °C cooling water for fluorescence-imaging analysis. To analyze turnover of dSpCas9-sgRNA and its variants, we preassembled the dSpCas9-sgRNA-DNA complex by incubating 0.5 pmol dSpCas9, 0.5 pmol 20-nt sgRNA or sgRNA variants, and 0.1 pmol target dsDNA for 1 h. We then added 0.5 pmol SpCas9-20-nt sgRNA into the reaction solution to cleave unbound DNA or dissociated DNA from preassembled dSpCas9-sgRNA-DNA for 6 h and 24 h at 37 °C. The reaction was quenched by the addition of 2 μL of denatured loading dye and the cleaved DNA resolved by 2% agarose gel electrophoresis.

The fluorescence imaging of gel electrophoresis was captured by Licor Odyssey infrared scanner and quantified by ImageJ. As both ends of target DNA substrates were labeled, the percentages of cut DNA or unbound DNA were calculated as the ratios of the combined intensity of two cut DNA bands to the combined intensity of total uncut and cut DNA or the ratios of the intensity of unbound DNA bands to the combined intensity of total bound and unbound DNA, respectively.

GFP⁺ reporter cells were transiently transfected with 0.25 μg each of dCas9 and sgRNA expression plasmids in 24-well plates. Cells were analyzed at 96 h post-transfection for GFP fluorescence intensity using Beckman Coulter CytExpert 2.0 normalized with mCherry transfection efficiency (TE). The GFP fluorescence intensity of cells transfected with each dCas9-sgRNA was calculated as below:\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$I\ \left(\mathrm{sgRNA}\right)=\frac{I\ \left(\mathrm{sgRNA}\mathrm{measured}\right)-I\ \left(\mathrm{CTRLmeasured}\right)\times \left(1-\mathrm{TE}\right)}{\mathrm{TE}}$$\end{document}IsgRNA=IsgRNAmeasured-ICTRLmeasured×1-TETEwhere I (sgRNA): GFP intensity of cells expressing dCas9-sgRNA; I (sgRNAmeasured): GFP intensity of cells after transfecting with dCas9-sgRNA; and I (CTRLmeasured): GFP intensity of cells after transfecting with dCas9-control sgRNA.

HEK293 cells were transiently transfected with GFP-P2A-Luciferase-based NHEJ reporter plasmids together with the expression plasmids for I-SceI or Cas9-sgRNA. The reporter was supplied at 0.025 μg in each well of 24-well plates. At 48 h post-transfection, cells were harvested and analyzed with the Dual Luciferase Reporter Assay system (Promega). All assays were done in triplicates and all values normalized for transfection efficiency against Renilla luciferase activities as internal control.

For analysis of targeted genome editing at endogenous genome loci, cells were collected after NHEJ was induced by Cas9-sgRNAs. Genomic DNA (gDNA) was isolated from these cells using a gDNA purification kit (Axygen). The targeted regions were PCR-amplified with Taq DNA polymerase (TsingKe Biological Technology). Respective primers for PCR are listed in Additional file 6: Table S5. The Illumina deep sequencing was performed at Novogene Co. Ltd, and subsequent data analysis was performed as previously described [28].

Potential off-target sites were identified using the latest version of the CRISPR Off-Target prediction website (http://crispor.tefor.net/↗). All potential sites were ranked by an off-target hit score, and high-ranked potential sites were selected. Off-target sites were amplified by PCR with primers listed in Additional file 6: Table S5 after gDNA extraction from cells transfected with Cas9-sgRNA at 3 days post-transfection. Off-target editing efficiency was determined by Illumina deep sequencing. The off-target rate was determined as the ratio of off-target to on-target mutagenesis levels.

HDR reporter mESC were transfected with Cas9-sgRNA and harvested 2 days post-transfection. For HDR reporter U2OS cells, 0.008 μg of the SV40 LT plasmid in 0.8 μg of total DNA was simultaneously transfected to initiate replication. gDNA was collected and the palindromic DNA sequences were amplified by touchdown PCR with primers listed in Additional file 6: Table S5. PCR amplicons were subcloned into CE Entry vector (Vazyme C114-02) and analyzed by Sanger sequencing. Deep sequencing of PCR amplicons was also performed, and their repair junctions were characterized by bioinformatics analysis.

Two-tailed Student's paired or unpaired t test was used for statistical analysis of repair frequencies, i.e., the frequencies of Cas9- and I-SceI-induced GFP⁺ cells, Cas9-induced GFP^– cells or Cas9-induced indels. Two-tailed Student's unpaired t test also allowed statistical analysis of comparison between two groups of sgRNAs targeting template strand of transcription or non-template strand, respectively. One-way ANOVA with post hoc Dunnett's multiple comparison test was performed for fold change of NHEJ alteration and HDR stimulation by inactivation of c-NHEJ between Cas9-sgRNA variants and their respective SpCas9-sgRNA controls and for fold change of off-target effect between NU7441 and DMSO, and between Xrcc4^+/+ and Xrcc4^–/– cells. Correlation between transcription silencing and the NHEJ increase was determined by linear regression analysis.

Additional file 1: Figure S1. Generation of DNA-PKcs^–/– and Ku80^–/– HDR reporter mESC clones. Figure S2. The effect of DNA-PKcs inhibition on the patterns of Cas9-induced NHEJ analyzed by targeted PCR amplicon Illumina sequencing. Figure S3. The effect of DNA-PKcs inhibition on the patterns of Cas9-induced NHEJ at the Cola1 and Rosa26 locus. Figure S4. Effect of DNA-PKcs inactivation on I-SceI-induced mutNHEJ. Figure S5. Sequences of mismatched or truncated sgRNAs used for weakening target interaction of Cas9-sgRNA. Figure S6. Effect of DNA-PKcs inhibition on SpCas9-mediated gene knockout (KO) at GFP gene. Figure S7. Replication locally disengages c-NHEJ at Cas9-induced DSBs. Figure S8. Analysis of palindromic sister chromatid ligation. Figure S9. Junctions of palindromic sister chromatid NHEJ products.Additional file 2: Table S1. Junction sequences of sister chromatid fusion by Sanger sequencing.Additional file 3: Table S2. Junction sequences of sister chromatid fusion products in mESC by PCR targeted amplicon sequencing.Additional file 4: Table S3. Junction sequences of sister chromatid fusion products in U2OS cells by PCR targeted amplicon sequencing.Additional file 5: Table S4. List of sgRNAs used in this study.Additional file 6: Table S5. List of PCR primers used in this study.Additional file 7. Review history.