Fig. 3: G1-phase DSB induction enhances SSA-mediated gene targeting.
From: Characterization and regulation of cell cycle-independent noncanonical gene targeting
a Schematic diagram of Cas9-mediated DNA cleavage at the HPRT gene using exon 3-targeting single-guide RNA (sgRNA). PAM, protospacer-adjacent motif. b Impact of Cas9-induced cleavage on TI frequency. Cells were transfected with p8.9HPRT-2A-Puro or p8.9HPRT-2A-Puro-95-95 along with pX330-Cas9-HPRT-Ex3 or pX330-U6-Chimeric_BB-CBh-hSpCas9. The ratio of TI frequency with DSB induction (Cas9+) to that without induction (Cas9-) is indicated. Data shown are the mean ± s.d. (n = 3). c Impact of Rad51 and Rad52 inhibition on Cas9-induced TI. Msh2-deficient (-) and proficient (+) Nalm-6 cell lines were subjected to co-transfection of targeting vector and pX330-Cas9-HPRT-Ex3, and treated with DOPA or B02 prior to replating. Data shown are the mean ± s.d. (n = 3). d Schematic representation of cell cycle-dependent Cas9 expression vectors. Expression patterns of each Cas9 protein inferred from the Fucci system are also indicated. PCBh, CMV enhancer/chicken β-actin hybrid promoter; NLS, nuclear localization signal. e Impact of cell cycle-regulated Cas9 expression on chromosomal HR repair. The sequence of SceGFP-targeting sgRNA is indicated, along with PAM and the I-SceI site. Data shown are the mean ± s.d. (n = 5). f Impact of cell cycle-regulated Cas9 expression on chromosomal SSA repair. The sequence of 3’GFP-targeting sgRNA is indicated, along with PAM and the I-SceI site. Data shown are the mean ± s.d. (n = 5). g Impact of targeted DSB induction on TI frequency. The TI frequency of p8.9HPRT-2A-Puro or p8.9HPRT-2A-Puro-95-95 without Cas9 in each cell line was taken as 1, and the relative TI frequency was calculated. Data shown are the mean ± s.d. (n = 4). h Impact of targeted DSB induction on TI frequency in HT1080 cells. The TI frequency of p8.9HPRT-2A-Bsr or p8.9HPRT-2A-Bsr-95-95 without Cas9 was taken as 1 in each cell line, and the relative TI frequency was calculated. Data shown are the mean ± s.d. (n = 3). Statistical significance in (b, c, g, and h) was determined by two-sided Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; NS, not significant. Source data are provided as a Source Data file.
