Fig 1: Both CAS9 and XCAS9 impair NHEJ and induce genetic mutations. (A and B) Expression of CAS9 and XCAS9 impairs NHEJ. Traffic Light Reporter system was established in 293 cells harboring the CAS9 and XCAS9 inducible expression vectors. After the induction of CAS9 and XCAS9 expression with 2 µg/mL doxycycline for 3 days (left panel), the efficiency of NHEJ (mcherry) and HDR (GFP) was analyzed by flow cytometry (middle panel). Statistic analysis of the efficiency of NHEJ (left panel of A) and HDR (B) is presented. n = 3. Data are presented as mean values ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. ns, non-significant. (C) The expression of CAS9 in hESCs induces genomic mutations at the endogenous HPRT locus. After hESCs harboring CAS9 inducible expression vector were selected with HAT medium for 5 days, they were treated with 2 µg/mL doxycycline for CAS9 expression for various time periods, and subsequently, treated with 5 µg/mL 6-TG or mock treated for 4 days. Mutational rate is calculated as the ratio of colony number in 6-TG treated samples versus untreated controls. n = 3. Data are presented as mean values ± SD. ***P < 0.001. (D) XCAS9 interacts with KU86. Protein extracts from 293FT cells expressing Flag-tagged CAS9 or XCAS9 were immunoprecipitated with anti-Flag antibody. The immune precipitates were analyzed for the presence of CAS9, XCAS9 and KU86. The relative ratio of KU86 versus CAS9 or XCAS9 is indicated. (E) The expression of XCAS9 increases the number of ?H2AX foci in hESCs. hESCs harboring XCAS9 inducible expression vector were treated with or without 2 µg/mL doxycycline for 3 days. n = 20. Scale bar, 10 µm. Data are presented as mean values ± SD. ***P < 0.01
Fig 2: KU promotes MRE11 binding to nascent DNA in BRCA-deficient cells, but suppresses binding of EXO1.a–d SIRF experiments showing that binding of PARP14 (a, b) and MRE11 (c, d) to nascent DNA in BRCA2-knockout HeLa (a, c) and DLD1 (b, d) cells is suppressed by depletion of KU70 or KU80. e EXO1 SIRF experiment showing that EXO1 is not recruited to nascent DNA in HeLa-BRCA2KO cells upon HU treatment. However, depletion of KU70 results in binding of EXO1 to nascent DNA under these conditions. ZRANB3 co-depletion suppresses this binding. EXO1 co-depletion reduces the signal, showing the specificity of the EXO1 SIRF readout. Single antibody controls are shown in Supplementary Fig. 6l. f–h EXO1 SIRF experiments showing binding of EXO1 to nascent DNA in DLD1-BRCA2KO (f) and HeLa-BRCA2KO (g, h) when the EdU labeling time was increased from 10 mins to 30 mins. EXO1 co-depletion reduces the signal, showing the specificity of the EXO1 SIRF readout. For all panels, at least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. i Schematic representation of the proposed model. KU binds the exposed DSB end of symmetrical reversed forks, protecting it against EXO1. At the same time, KU bound on the reversed fork recruits the PARP14-MRE11 complex, and through its endonuclease activity MRE11 creates a nick, which is then process by its 3’ to 5’ exonuclease activity towards the DSB end. This results in release of KU from the DSB end. In BRCA-proficient cells, loading of RAD51 stabilizes the ssDNA overhang against further nucleolytic processing. In the absence of KU, EXO1 engages the DSB end with its 5’ to 3’ exonuclease activity for, but loading of RAD51 by the BRCA pathway on the partially resected DSB end stabilizes it against further degradation. In BRCA-deficient cells, the partially resected structure is susceptible to continuous (long-range) degradation by EXO1 on the 5’ end strand, and MRE11 (and potentially other 3’ to 5’ exonucleases) on the 3’ end strand. Created with BioRender.com. Source data are provided as a Source Data file.
Fig 3: dCAS9 and Cpf1 impair NHEJ and induce genetic mutations. (A) Co-immunoprecipitation assay confirmed the interaction between dCAS9 and KU86. (B) Comet assay analysis of DNA damage in hESCs expressing dCAS9 or treated with doxorubicin. CTL, human fibroblasts with lentiviral empty vector were treated with 2 µg/mL doxycycline for three days; Doxy, Dox, Doxy + Dox, human fibroblasts with lentiviral CAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days or 0.5 µmol/L Dox for 2 h or 2 µg/mL doxycycline for three days + 0.5 µmol/L Dox for 2 h, respectively. Tail length was analyzed using Image J software. Data are represented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001. (C) The expression of dCAS9 induces mutation of endogenous HPRT gene. WT, WT hESCs; CTL, CAS9, dCAS9, hESCs with empty expression vector, CAS9 inducible expression vector. Cells with dCAS9 inducible expression vector were treated with 2 µg/mL doxycycline for 3 days before HAT treatment. n = 3. Data are presented as mean value ± SD. **P < 0.01, ***P < 0.001. (D) The expression of Cpf1 increased the levels of ?H2AX. (E) Cpf1 interacts with KU86 as confirmed by Co-immunoprecipitation. Protein extract of Flag-tagged Cpf1 was immunoprecipitated with anti-Flag antibody and the presence of Cpf1 and KU86 in the immunoprecipitate was examined by Western blot
Fig 4: Differential regulation of nascent strand degradation by EXO1 and MRE11 by the KU complex.a, b PARP14-KU80 PLA experiments in HeLa cells showing that the interaction between PARP14 and KU is increased by BRCA2 deficiency. Representative micrographs, with scale bars representing 10 µm (a) and quantifications (b) are shown. The specificity of the readout is demonstrated by the loss of PARP14-KU80 PLA foci in HeLa-PARP14KO cells (a). At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). c, d KU SIRF experiments showing that KU70 (c) and KU80 (d) binding to nascent DNA is increased by HU treatment and BRCA2 deficiency. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Validation of the KU SIRF readout is shown in Supplementary Fig. 6b–d. e, f KU SIRF experiments showing that KU70 (e) and KU80 (f) binding to nascent DNA upon HU treatment in HeLa-BRCA2KO cells is suppressed by knockdown of fork reversal factors ZRANB3, SMARCAL1 and PARP1. At least 100 cells were quantified for each condition. Bars indicate the mean values, error bars represent standard error of the mean, and asterisks indicate statistical significance (t-test, two-tailed, unpaired). Schematic representations of the assay conditions are shown at the top. Western blots confirming the depletions are shown in Supplementary Fig. 6e. g, h DNA fiber combing assays showing that knockdown of KU70 (g) or KU80 (h), with two different siRNA oligonucleotides for each, does not impact HU-induced fork degradation in HeLa-BRCA2KO cells. Western blots confirming KU70 and KU80 depletion are shown in Supplementary Fig. 6f. i DNA fiber combing assays showing that KU80 co-depletion restores fork degradation in PARP14-depleted HeLa-BRCA2KO cells. Western blots showing co-depletion of PARP14 and KU80 are presented in Supplementary Fig. 6g. j DNA fiber combing assays showing that KU80 depletion restores fork degradation in HeLa-BRCA2KOPARP14KO double knockout cells, and this degradation is not performed by MRE11 since it is not rescued by mirin treatment. k, l DNA fiber combing assays showing that KU80 co-depletion restores fork degradation in PARP14-depleted BRCA2-knockout cells, and this degradation is not performed by MRE11 since it is not rescued by mirin treatment. Co-depletion of EXO1, but not inhibition of MRE11 by mirin, restores fork protection in KU80-depleted BRCA2-knockout cells. Similar results were obtained in HeLa-BRCA2KO (k) and DLD1-BRCA2KO (l) cells. Western blots showing co-depletion of EXO1 and KU80 are presented in Supplementary Fig. 6h. m DNA fiber combing assays showing that EXO1 knockout restores fork protection in KU80-depleted HeLa-BRCA2KO cells. Two independent BRCA2KOEXO1KO double knockout clones were analyzed. Western blots confirming the EXO1 and BRCA2 knockout are shown in Supplementary Fig. 6i. For panels g–m, the ratio of CldU to IdU tract lengths is presented, with the median values marked on the graphs and listed at the top. At least 100 tracts were quantified for each sample. Asterisks indicate statistical significance (Mann-Whitney, two-tailed). Schematic representations of the assay conditions are shown at the top. Source data are provided as a Source Data file.
Fig 5: CAS9 interacts with KU86 and disrupts the formation of DNA-PK. (A and B) Reciprocal immunoprecipitation shows the interaction between CAS9 and KU86. Protein extracts from hESCs expressing CAS9 were immunoprecipitated with anti-KU86 (A) or anti-CAS9 (B), and immune precipitates were analyzed for the presence of KU86 and CAS9. (C) The interaction between CAS9 and KU86 was confirmed by the proximity ligation analysis (PLA). Cell nucleus were revealed by DAPI (Blue) staining and the CAS9-KU86 interaction indicated by red color. Scale bar, 25 µm. Unpaired t test. n = 20. Data are presented as mean value ± SD. ***P < 0.001. (D) Mapping the domain of CAS9 involved in the interaction with KU86. The Flag-tagged deletional mutants of CAS9 expressed in 293FT cells were immunoprecipitated with anti-flag antibody. Immune precipitates were analyzed for the presence of CAS9 mutants and KU86. (E) The expression of the PAM domain (1100) of CAS9 disrupted the interaction between CAS9 and KU86. The levels of CAS9, KU86, PAM in the input and immunoprecipitate were analyzed by Western blot. The ratio of CAS9 versus KU86 in the immunoprecipitate is shown at the bottom. (F) CAS9 disrupts the formation of DNA-PK complex. Protein extracts of cells in the presence and absence of CAS9 and DOX treatment were immunoprecipitated with anti-KU86 antibody. The levels of KU70, DNA-PKcs and CAS9 in the immunoprecipitate were analyzed. The relative ratios of DNA-PKcs versus KU86 or KU70 versus KU86 are indicated
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