Fig. 5
From: Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair
Analyzing the expression levels of genes in S-G2 cell cycle phases and HDR pathway by qPCR and WB. A qPCR assay showing the main factors controlling S and G2 progression (CDK1, CCNB1 and CCNA2) and HDR factors (CTIP, RPA1 and RPA2) exhibited greatly increased expression in 293T cells with IRI and MITO treatment compared to DMSO treatment control, whereas their up-regulation was less or insignificant in DOC and NOC treatment groups. Data are mean ± SD from 3 independent samples. B WB results showing similar gene expression changes as qPCR. CDK1 and RPA2 showing evident phosphorylation in all small molecule treated cells. C The data indicate cell cycle synchronization increases CDK1, CTIP and RPA2 activity to promote HDR. The small molecule inhibitors induce up-regulated CDK1 expression or activity, which promotes efficient DSB end resection by phosphorylating CITP and other nucleases and thus prevents DNA repair by NHEJ. Efficient break end resection generates sufficient ssDNA overhangs for RPA complex coating which is essential for HDR initiation