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Fig. 7 | Genome Biology

Fig. 7

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

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-gEJW10 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-gHRC2 (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-gHRC4, 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

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