Inactivation of c-NHEJ induces varying stimulation of Cas9-induced HDR among targets
Like any other DSBs, Cas9-induced DSBs are repaired by c-NHEJ, a-NHEJ and HDR (Fig 1a). Thus, inactivation of the predominant NHEJ pathway c-NHEJ is expected to channel more Cas9-induced DSBs towards HDR for repair, increasing the usage of HDR [20–22] (Fig 1a). If target interaction of Cas9-sgRNA influences DSB repair pathway choice after DNA cleavage at its targets, the involvement of c-NHEJ in repair of Cas9-induced DSBs would change between targets with different target interaction for Cas9-sgRNA. Inactivation of c-NHEJ would thus lead to varying degrees of HDR stimulation at these sites. To test this hypothesis, we used Streptococcus pyogenes Cas9 (SpCas9) in complex with its sgRNA partner (Cas9-sgRNA) to induce site-specific DSBs at different sites in a single-copy HDR reporter integrated at the Rosa26 locus in the genome of mouse embryonic stem cells (mESC) and analyzed the impact of c-NHEJ inactivation on Cas9-induced HDR (Fig 1a). This HDR reporter contains two inactivated GFP copies, TrGFP truncated at the 5’-end and I-SceI-GFP interrupted with an 18-bp recognition site for the rare cutting endonuclease I-SceI . Using TrGFP of the sister chromatid as a template, HDR of a site-specific chromosomal DSB induced by I-SceI or CRISPR nucleases generates a wild-type GFP copy and thereby GFP+ cells (Fig 1a). The frequency of GFP+ cells induced by I-SceI or CRISPR nucleases reflects the level of HDR. Like I-SceI-induced HDR, Cas9-induced HDR was increased by NU7441 at the sites targeted by gHRC1, gHRC2 and gHRC3 and the extent of this stimulation was different among these three targets (Fig 1b). Surprisingly, DNA-PKcs inhibition did not elevate HDR induced by Cas9-gHRC4 and Cas9-gHRC5 (Fig 1b), suggesting a possibility of little c-NHEJ involvement in DSB repair at either the gHRC4 site or the gHRC5 site. We also used CRISPR/Cas9 gene editing to generate isogenic wild-type, DNA-PKcs–/– and Ku80–/– mESC clones containing the HDR reporter (Additional file 1, Fig S1a, b). Using one of these clones, along with isogenic XRCC4+/+ and XRCC4–/– HDR reporter mESC previously established , we found that deletion of DNA-PKcs, Ku80 orXRCC4 significantly enhanced HDR induced by gHRC1, gHRC2 or gHRC3 in complex with SpCas9, as well as HDR induced by I-SceI (Fig 1c and Additional file 1, Fig S1c). However, deletion of DNA-PKcs or Ku80 stimulated no HDR at the gHRC4 and gHRC5 sites whereas deletion of XRCC4 caused limited degrees of HDR stimulation at these two sites (Fig 1c). Therefore, the extents of HDR stimulation by c-NHEJ inactivation varied among these five different targets from little stimulation at the gHRC4 and gHRC5 sites to stimulation by 90.7% at the gHRC2 target (Fig 1c). It is possible that c-NHEJ is engaged to different extents among targets where Cas9-induced HDR is stimulated to varying degrees by inactivation of c-NHEJ, and not even engaged at all at the targets where Cas9-induced HDR is not stimulated by inactivation of c-NHEJ.
Repair of Cas9-induced DSBs involves c-NHEJ to varying degrees at different targets
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  and analyzed the effect of c-NHEJ inactivation on the frequencies of Cas9-induced insertion or deletion mutations (indels) (Fig 1d). 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 . 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-NHEJ can generate indels at the repair junction. In theory, only a third of indels can lead to GFP+ cells, and the frequency of Cas9-induced GFP+ cells thus represents the relative efficiency of Cas9-induced indels  (Fig 1d). As c-NHEJ and a-NHEJ generate different proportions of accurate NHEJ (accNHEJ) products and indel-based mutagenic NHEJ (mutNHEJ) products , inactivation of c-NHEJ would channel more Cas9-induced DSBs towards error-prone a-NHEJ in addition to HDR, altering 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 gEJW3 or gEJW7, 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 gEJC5, gEJW4, gEJW5 and gEJW6 to different extents, varying from 16.6% to 69.2% (Fig 1e). This indicates that the participation of c-NHEJ varies in repair of Cas9-induced DSBs at different targets.
Additionally, using targeted PCR amplicon deep sequencing as done before , we 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). 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.
Target recleavage by Cas9 amplifies the mutagenicity of c-NHEJ
Like I-SceI, CRISPR nucleases generate DSBs with directly ligatable ends. Previous studies have demonstrated that c-NHEJ is intrinsically accurate for these ends [5,6,26]. 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 . Thus, inactivation of c-NHEJ would increase the use of a-NHEJ in each round of CRISPR/Cas9 genome editing. Since a-NHEJ 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, Fig S2a). In consistent with previous findings that inactivation of c-NHEJ stimulates production of I-SceI-induced GFP+ cells [25,27], inhibition of c-NHEJ with NU7441 increases I-SceI-induced GFP+ cells by more than 2-fold (Additional file 1, Fig S2b, 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 wondered what the difference is. While c-NHEJ of both I-SceI- and Cas9-induced DSBs generates a significant level of accurate end-joining products in each round of repair at their respective targets, regenerating the target sites for recleavage, the recelavage by Cas9 may be much more efficient than I-SceI [28,29]. Thus, in cells expressing abundant Cas9-sgRNA, these target sites could be efficiently recleaved and repaired until indels are introduced and accumulated (Fig 2a). As a result, c-NHEJ appeared mostly mutagenic for Cas9-induced DSBs and inactivation of c-NHEJ would reduce Cas9-induced indels (Fig 1e). To test this possibility, we reduced the transfection amount of Cas9 or sgRNA into the NHEJ reporter mESC to limit the Cas9 recleavage in the cells and determined whether Cas9-induced GFP+ cells would be stimulated by DNA-PKcs inhibition after Cas9 recleavage is restricted (Fig 2a). We found that overall Cas9-induced GFP+ cells was reduced with a low amount of Cas9-gEJW6 in the absence of c-NHEJ inhibition (Fig 2b). This could be explained by either less initial Cas9 cutting, less Cas9 recleavage of accurate repair products or both. While NU7441 suppressed production of GFP+ cells induced by a high amount of Cas9-gEJW6 at 0.25μg each, the inhibitor started to stimulate production of GFP+ cells when the amount of Cas9 and gEJW6 was both reduced to 0.001μg (Fig 2b, c). In contrast, at the gEJW7 target, NU7441 did not alter the frequency of GFP+ cells induced by Cas9 and gEJW7 at an amount ranging from 0.25μg to 0.0001μg (Fig 2d, e). This further confirms that c-NHEJ is not involved in repair of Cas9-induced DSB at the gEJW7 target after the interference of target recleavage is minimized.
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 gEJW4 and gEJW6 with the transfection amount of Cas9-sgRNA at 0.001μg, Cas9-induced indels were also reduced as expected (Fig 2f). DNA-PKcs inhibition and XRCC4 deletion did not suppress production of Cas9-induced GFP+ cells any more or even reversed to stimulation at the gEJW4 and gEJW6 targets but remained to exert no effect on the level of Cas9-induced GFP+ cells at the gEJW3 or gEJW7 site (Fig 2f). In fact, XRCC4 deletion elevated the frequency of Cas9-induced GFP+ cells by 59.6 ± 14.2% (P<0.05) at the gEJW4 target and 81.5 ± 24.5% (P<0.05) at the gEJW6 target with 0.001μg of Cas9-sgRNA (Fig 2f), a reverse from reduction of Cas9-induced GFP+ cells by 58.1 ±3% and 60.4 ± 2.4% respectively at these two targets with 0.25μg 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.
Differently, at the gEJC5 or gEJW5 target, with the transfection amount of Cas9-sgRNA at 0.001μg, DNA-PKcs inhibition and XRCC4 deletion still inhibited the generation of Cas9-induced GFP+ cells; but this inhibition was reduced (Fig 2f). At the gEJC5 target, XRCC4 deletion reduced Cas9-induced GFP+ cells by 37.0 ±3.2% (P<0.001) with 0.001μg of Cas9-sgRNA, a smaller reduction than 55.8 ± 2.6% (P<0.001) with 0.25μg of Cas9-sgRNA (Fig 2f vs. 1e). At the gEJW5 target, this reduction of GFP+ cells by XRCC4 deletion is 57.6 ± 7.5% (P<0.001) with 0.001μg of Cas9-sgRNA but 69.2 ±1.5% (P<0.01) with 0.25μg of Cas9-sgRNA (Fig 2f vs. 1e). This suggests that Cas9 recleavage could still abrogate the stimulatory effect of c-NHEJ inactivation on Cas9-induced indels at the gEJC5 or gEJW5 target sites where limiting Cas9 recleavage does not fully abolish the suppression of Cas9-induced indels by c-NHEJ inactivation. Similar to the gEJW7 target, no effect by c-NHEJ inactivation was detected at the gEJW3 target with neither 0.001μg nor 0.25μg of Cas9-sgRNA (Fig 2f and Fig 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, 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.
Weakening target interaction of Cas9-sgRNA biases repair of Cas9-induced DSBs towards c-NHEJ
To further determine whether c-NHEJ repair of Cas9-induced DSBs is influenced by target interaction of Cas9-sgRNA, we compare the c-NHEJ engagement at the same target by changing the interaction between Cas9-sgRNA and target DNA. In this setting, the effects of DNA sequences or chromatin structures are fixed and only target interaction is allowed to change. We mutated either sgRNA or SpCas9 for two sites targeted by gEJC5 and gEJW7 in the NHEJ reporter to reduce Cas9-sgRNA target interaction. In consistent with previous observation that reducing Cas9-sgRNA target interaction generally lowered the efficiency of genome editing [30–33], induction of Cas9-induced GFP+ cells was less efficient with mismatched or truncated sgRNA variants (i.e. the C2A mismatch, the T15A mismatch and the truncated 16nt for gEJC5, and A1T, A4C and T15A for gEJW7) and with SpCas9 variants eSpCas9 and SpCas9-HF1, both of which were engineered to have less target interaction (i.e. lower binding affinity) and higher specificity to target DNA (Fig 3a). The sequences of the sgRNA variants are listed in Additional file 1, Fig S3a. As in Fig 1e, DNA-PKcs inhibition and XRCC4 deletion reduced Cas9-induced GFP+ cells respectively by 30.1% and 62.4% at the site targeted with SpCas9-gEJC5, again suggesting significant DNA recleavage by Cas9 (Fig 3a). In contrast, at the same target, the gEJC5 variants C2A and T15A alleviated or even reversed this NU7441-mediated reduction, and the gEJC5 variant 16nt and SpCas9-HF1 strongly reversed the reduction by XRCC4 deletion as the fold changes of NHEJ stimulation induced by DNA-PKcs inhibition or XRCC4 deletion between these Cas9-sgRNA variants and the SpCas9-gEJC5 20nt control were more than 1 and up to 5.1 (Fig 3a). At the site targeted by gEJW7, neither DNA-PKcs inhibition nor XRCC4 deletion had effect on the frequency of Cas9-induced GFP+ cells as shown in Fig 1a (Fig 3a), indicating no engagement of c-NHEJ at this site. However, the gEJW7 mismatch variant T15A and SpCas9-HF1 allowed significant NU7441-mediated stimulation of Cas9-induced GFP+ cells (Fig 3a). T15A, eSpCas9 and SpCas9-HF1 also elicited stimulatory effect of XRCC4 deletion on Cas9-induced GFP+ cells at the gEJW7 target site as the fold changes of this NHEJ stimulation between the Cas9-sgRNA variants and the SpCas9-gEJW7 20nt control were up to 3.5-fold (Fig 3a). This suggests that in repair of Cas9-induced DSBs, the weaker the Cas9-sgRNA target interaction is, the more preferentially c-NHEJ is engaged.
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 S3b). 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 S3b). 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 toward c-NHEJ in repair of Cas9-induced DSBs.
Weakening target interaction of Cas9-sgRNA enhances stimulatory effect of c-NHEJ inactivation on Cas9-induced HDR
Consistently with previous studies [20–22], inactivation of c-NHEJ stimulates HDR induced by CRISPR nucleases as well as I-SceI (Fig 1b, c and Additional file 1, Fig S1c). 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 gHRC4, and A1T, C2A and 17nt for gHRC2) and SpCas9 variants eSpCas9, SpCas9-HF1 and xCas9 (i.e. xCas9-3.7), except eSpCas9-gHRC4, SpCas9-gHRC2 17nt and xCas9-gHRC2 20nt (Fig 3d, e and Additional file 1, Fig S3c).
At the site targeted by gHRC4, 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 gHRC4 G1C and 17nt, as well as with SpCas9-HF1 (Fig 3d). In addition, XRCC4 deletion stimulated Cas9-induced HDR with the gHRC4 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 gHRC2, 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-gHRC2 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-gHRC4 20nt, SpCas9-gHRC2 A1T, SpCas9-gHRC2 17nt or xCas9-gHRC2 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, providing the basis for the enhanced stimulatory effect of c-NHEJ inactivation on Cas9-induced HDR.
Inactivation of c-NHEJ increases off-target activity of CRISPR/Cas9
As mismatches in base pairing between sgRNA and off-target sites weaken the interaction of Cas9-sgRNA with off-target sites, it is anticipated that c-NHEJ would be engaged proportionally more at off-target sites than at on-target sites. In addition, target recleavage occurs less at off-target sites. Thus, inactivation of c-NHEJ would increase the engagement of a-NHEJ at off-target sites. As a-NHEJ is more error prone even for directly ligatable ends, inactivation of c-NHEJ leads to proportionally more mutNHEJ events and exacerbates off-target effects in CRISPR/Cas9 genome editing. To test this hypothesis, we analyzed the effects of DNA-PKcs inhibition and XRCC4 deletion on off-target activities of Cas9 at 7 potential off-target sites for gPnpla3 and 6 potential off-target sites for gMertk and calculated the fold change of off-target effect due to DNA-PKcs inhibition and XRCC4 deletion. We found that both NU7441 and XRCC4 deletion slightly reduced on-target editing by Cas9-gPnpla3 and Cas9-gMertk by about 15-21%, suggesting significant on-target DNA recleavage. In contrast, the frequencies of Cas9-induced indels at off-target sites were not reduced by either DNA-PKcs inhibition or XRCC4 deletion, but increased at many of these sites (Fig 4a, b, left). The fold change of off-target effect was more than 1 and even over 2 at some sites by c-NHEJ inactivation (Fig 4a, b, right). This suggests that inactivation of c-NHEJ aggravate off-target effect in CRISPR/Cas9 genome editing.
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 [34–39]. Given that DNA-PKcs inhibition by NU7441 stimulated Cas9-induced HDR in the HDR reporter at the targets by gHRC1 and gHRC2 (Fig1b), we also performed off-target analysis for 6 potential off-target sites for Cas9-gHRC1 and Cas9-gHRC2 respectively. After NU7441 treatment, the frequencies of on-target indels induced by Cas9-gHRC1 and Cas9-gHRC2 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 4c, d, left), and the fold change of off-target effect was elevated up to 2.5 (Fig 4c, d, right). This again suggests that both chemical inhibition and genetic inactivation of c-NHEJ exacerbate off-target effects in CRISPR/Cas9 genome editing.
Local transcription does not affect involvement of c-NHEJ in repair of Cas9-induced DSBs
As weakening target interaction of Cas9-sgRNA promotes c-NHEJ engagement in repair of Cas9-induced DSBs, we wondered how the strength of Cas9-sgRNA target interaction controls the extent of c-NHEJ engagement. Since the Cas9-sgRNA complex remains bound to its target after DNA cleavage due to persistent target interaction of Cas9-sgRNA, it is possible that DNA ends are buried in the complex and do not fully elicit the DNA damage response (DDR) or engage any repair pathways before DNA end exposure [10–15]. While some ends are exposed by spontaneous dissociation of Cas9-sgRNA from cleaved target DNA and readily engage c-NHEJ, the others may require local transcription machinery to dislodge the target-bound Cas9-sgRNA complex . The collision with local transcription machinery generates different DNA end configurations that may be unsuitable for binding c-NHEJ factors. If this was the case, we reasoned that the gene silencing activity (i.e. the transcription-blocking capability) of catalytically dead Cas9 (dCas9)-sgRNA at a given target would be negatively correlated with the extent of c-NHEJ participation in repair of Cas9-induced DSBs at the same site. Thus, using the single-copy GFP gene expression cassette integrated at the ROSA26 locus in the genome of mESC, we induced the GFP gene silencing at various sites by catalytically dead SpCas9 (dSpCas9) and also generated GFP– cells by SpCas9-induced GFP knock-out (KO) editing at these sites (Additional file 1, Fig S4a). We examined any potential correlation between dSpCas9-mediated gene silencing and c-NHEJ involvement in SpCas9-induced GFP KO at the same sites. While dSpCas9-sgRNA exhibited variable gene silencing activities at many of these sites (Fig 5a), the effect of DNA-PKcs inhibition on GFP KO varied from no effect for gGC1, gGC4, gGC7, gGC10, gGC14, gGC15 and gGW5 to about 4-fold stimulation for gGC9 and gGW2 among targets (Fig 5b and Additional file 1, Fig S4b). No apparent bias towards either template strand of transcription or non-template strand was detected in both transcription silencing by dSpCas9-sgRNA and DNA-PKcs involvement reflected by stimulation of SpCas9-induced GFP KO by NU7441 (Fig 5c). Importantly, no correlation was observed between transcription silencing by dSpCas9-sgRNA and stimulation of SpCas9-induced GFP KO by DNA-PKcs inhibition (Fig 5D; P=0.78), excluding the possibility that a collision with local transcription control the involvement of c-NHEJ in repair of Cas9-induced DSBs.
To further determine the effect of the collision between local transcription and Cas9-sgRNA on the engagement of c-NHEJ for Cas9-induced indels, we used catalytically dead Staphylococcus aureus Cas9 (dSaCas9)-sgRNA to block the translocating RNA polymerase (RNAP), preventing its collision with downstream site-specific DSBs induced by SpCas9 (Fig 5e). Among 6 sgRNAs tested for transcriptional blockage, only gSaGW1 and gSaGW2, 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 5e). The frequency of GFP– cells induced by SpCas9-gGC4 and SpCas9-gGW5 at a transfection amount of 0.125μg or 0.0005μg for each plasmid was not altered by DNA-PKcs inhibition with NU7441, and this non-effect was little changed by co-transfection with either dSaCas9-gSaGW1 or dSaCas9-gSaGW2 (Fig 5f). This suggests that transcription blockage by dSaCas9-sgRNA (e.g. dSaCas9-gSaGW1 and dSaCas9-gSaGW2) would not affect the extent of c-NHEJ engagement in repair of SpCas9-induced DSBs and further confirms that a collision with local transcription do not control c-NHEJ engagement in repair of Cas9-induced DSBs.
Local replication abolishes c-NHEJ engagement at Cas9-induced DSBs
Like transcription, local DNA replication could also collide with Cas9-sgRNA that remains bound to the cleaved target and dislodge Cas9-sgRNA from the cleaved DNA, generating end configurations that may not be suitable for engaging c-NHEJ. Additionally, the collision with the replication fork occurs in S phase, where HDR is favored for replication-coupled DSB repair. Thus, to investigate whether collision with local DNA replication underlies the biased disengagement of c-NHEJ in repair of Cas9-induced DSBs at some target sites, we transfected HEK293 cells with a plasmid containing an SV40 origin-ATG-GFP-P2A-FLuc NHEJ reporter cassette, together with expression plasmids for SV40 large T antigen (LT), I-SceI or the SpCas9-gEJW10 complex, and the Renilla luciferase (RLuc) gene as internal control. The expression of SV40 LT drives bidirectional DNA replication via the SV40 origin, and the expression of I-SceI or SpCas9-gEJW10 induces a site-specific DSB between the “Koz-ATG” and the “ATG-GFP-P2A-FLuc” (Fig 6a). Repair of I-SceI- or Cas9-induced DSBs mostly by c-NHEJ generate indels that can proportionally reframe the originally out-of-frame firefly luciferase (FLuc) gene in the NHEJ reporter plasmids to in-frame in the cells and induce synthesis of active firefly luciferase. The frequency of I-SceI- or Cas9-induced indels can thus be measured as a relative ratio of FLuc to RLuc by luminescence assays. Treatment with NU7441 reduced I-SceI-induced indels by 62.0±7.3% in this assay, but the level of this reduction was similar at 58.0±6.1% with the expression of SV40 LT (Fig 6a), suggesting little effect of local DNA replication on I-SceI-induced indels. However, while Cas9-induced indels was also suppressed by 83.0±2.3% with NU7441, DNA replication initiated by SV40 LT significantly attenuated this repressive effect to 33.0±5.8% (Fig 6a). This suggests that local DNA replication driven by SV40 LT might inhibit the involvement of c-NHEJ in repair of Cas9-induced DSBs.
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 6b), 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 gHRC1, gHRC2, gHRC3, gHRC4 and gHRC5 to different degrees, as well as by I-SceI (Fig 6b and Additional file 1, Fig S5a), 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-gHRC2, Cas9-gHRC3 and Cas9-gHRC4 were repressed in a gradual and dose-dependent manner (Fig 6b and Additional file 1, Fig S5b, 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 0.032μg (1/25 of total DNA transfected) for Cas9-gHRC4 and 0.16μg (1/5 of total DNA transfected) for Cas9-gHRC2 (Fig 6b and Additional file 1, Fig S5b, 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 toward 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 6c). 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-gHRC4 in mESC, we found the HDR bias was nearly 3-fold lower with gHRC4 variants (G1C and 17nt) than with gHRC4 (Fig 6d), indicating a reduced HDR preference when the interaction of SpCas9-sgRNA to its target is weakened. At the site targeted by gHRC2, 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 0.16μg (Fig 6b), SV40 LT expression at the same transfection amount increased the HDR bias by nearly 2-fold (Fig 6d), 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.
Palindromic fusion of sister chromatids arises from collision of Cas9-sgRNA at cleaved targets with DNA replication
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 6e). 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 [40–43] (Fig 6e). Because neither DNA-PKcs nor Ku80 is engaged at Cas9-induced DSBs at the gHRC4 target site for repair in the HDR reporter in mESC (Fig 1b, c), it is likely that Cas9-gHRC4 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 gHRC4 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 the interference in PCR amplification by palindromic DNA sequences . Given potentially asymmetric sequence deletion at the junction of SCF, 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-gHRC4 in the HDR reporter and detected PCR bands over 250bp in mESC (Fig 6f and Additional file 1, Fig S6a). 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-gHRC4 cleavage site (Fig 6f and Additional file 1, Fig S6a). This is consistent with the observation that DNA-PKcs inhibition stimulates HDR induced by Cas9-gHRC4 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 6g and Additional file 1, Fig S6b). They all contained some GFP sequences inverted around the break site but no palindromic GFP sequences, indicating that SCF may occur but palindromic sequences may be lost during repair or may not be amplified by PCR (Additional file 2, Table S1). In addition, 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 6g and Additional file 1, Fig S6b). 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. 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 S7a, 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.