Fig. 3: Dual inhibition of MMR and p53 improve PE outcome.
From: Enhancing genome editing in hPSCs through dual inhibition of DNA damage response and repair pathways
A Scheme of the prime editing process, the DNA single-strand break (SSB) response is colored in blue, mismatch repair (MMR) process is colored in red. Created with BioRender.com. B Image of crystal violet assay and relative areas (normalized by Mock) in H9-hESCs after transfection of Cas9 without gRNA (Mock), Cas9, PE2, and PE2 with p53DD vector (PE2 + DD). C Cell death after gene editing was tested by live-cell imaging and SYTOX staining. The imaging was started after 24 h of transfection of pegRNA for HEK4 ATCG insertion and PE4. D Prime editing efficiency for HEK3 CTT insertion by PE4 and PE5(Mock), PE4 and PE5 with p53DD expression (DD) in H9-hESCs (n = 3). E Scheme of PE4stem construction, created with BioRender.com. F Prime editing efficiency for indicated target by PE4max, PE4stem, PE5max, and PE5stem (n = 3 except the designated replicates). G, H Relative prime editing efficiency of PE4max and PE4stem (G) and PE5max and PE4stem (H), I Off-target editing of HEK3 1–5 deletion at off-target sites (OT1, OT2, OT3, OT4 and OT5) by PE4max and PE4stem. (n = 3). J Deletion more than 100 base pair long at RNF2 target treated with PE4max and PE4stem analyzed by Nanopore long-read sequencing (n = 3). K A jitter plot of the DNA and RNA base substitution from whole-exome sequencing and transcriptome data of PE4max and PE4stem treated cells with HEK3 CTT insertion pegRNA (n = 1). L Karyotype of unedited (RNF2 Mock and HEK3 Mock) and edited single clones (HEK3 #1, HEK3#2 and RNF1#1) of H9-hESCs (44 + XX). M The mRNA levels of pluripotency marker genes in hESCs, unedited (RNF2 Mock and HEK3 Mock) and edited clones (HEK3 #1, HEK3#2 and RNF1#1), human mesenchymal stem cells (hMSCs) as a negative control (n = 2). n always represents the biologically independent samples if not else described. Bars represent mean values, and error bars represent the S.D. of independent biological replicates. Detailed information on statistical analysis is listed in the “Statistical analysis” section. The source data of B–D, F–J, and M are provided in the Source Data file.
