Fig. 1: Programming key genes of HDR and NHEJ pathways enhanced HDR efficiency. | Cell Discovery

Fig. 1: Programming key genes of HDR and NHEJ pathways enhanced HDR efficiency.

From: Programmable DNA repair with CRISPRa/i enhanced homology-directed repair efficiency with a single Cas9

Fig. 1

a Diagram of dgRNA-MS2:MPH expression vector for activating key genes of HDR pathway. b Diagram of dgRNA-Com:CK expression vector for repressing key genes of NHEJ pathway. c Diagram of the TLR system. Cas9/sgRNA can induce DSBs in the target site. If DSBs are repaired by NHEJ, 3n + 2 bp frame shift indels can restore mCherry expression, which accounted for approximately 1/3 mutagenic NHEJ events. Alternatively, if DSBs were repaired according an intact EGFP template, the mutations in bf-Venus will be corrected, leading to Venus (EGFP variant) expression. d Quantitative results of HDR efficiency by programming essential components of DNA repair pathways (Vector vs. dgCDK1-2, p = 0.0014; Vector vs. dgCtIP-1, p = 0.0079; Vector vs. dgLIG4-1, p = 0.0031; Vector vs. dgKU70-2, p = 0.026; Vector vs. dgKU80-1, p = 0.0338; Vector vs. dgCDK1-2 + dgCtIP-1, p = 0.0138; Vector vs. dgCDK1-2 + dgLIG4-1, p = 0.0022; Vector vs. dgCDK1-2 + dgKU70-2, p = 0.075; Vector vs. dgCDK1-2 + dgKU80-1, p = 0.0493; Vector vs. dgCtIP-1 + dgLIG4-1, p = 0.0692; Vector vs. dgCtIP-1 + dgKU70-2, p = 0.0245; Vector vs. dgCtIP-1 + dgKU80-1, p = 0.0063; Vector vs. dgLIG4-1 + dgKU70-2, p = 0.0337; Vector vs. dgLIG4-1 + dgKU80-1, p = 0.0606; Vector vs. dgKU70-2 + dgKU80-1, p = 0.0299), the representative flow cytometry figures are shown in Fig. S3c. e Strategy for insertion of an EGFP reporter gene into the human AAVS1 locus using CRISPR-Cas9 in human cells. The SA-T2A-EGFP promoterless cassette was flanked by two AAVS1 homology arms, left arm (489 bp) and right arm (855 bp). SA, splice acceptor, T2A, a self-cleaving peptide, PA, a short polyA signal, primer F and primer R were designed for EGFP-positive events identification and sequencing. f Chromatogram and sequences of HDR-positive events. Partial donor sequences and adjacent genomic DNA sequence were represented. g–l HDR efficiency was determined in three different cell lines, HEK293 (Vector vs. dgCDK1-2, p = 0.0153; Vector vs. dgKU80-1, p = 0.0404; Vector vs. dgCDK1-2 + dgKU80-1, p = 0.029), HEK293T (Vector vs. dgCDK1-2, p = 0.0008; Vector vs. dgKU80-1, p = 0.0227; Vector vs. dgCDK1-2 + dgKU80-1, p = 0.0087) and HeLa (Vector vs. dgCDK1-2, p = 0.015; Vector vs. dgKU80-1, p = 0.0216; Vector vs. dgCDK1-2 + dgKU80-1, p = 0.0004). CDK1 activation and/or KU80 repression significantly increased HDR efficiency. These cell lines were co-transfected with SA-T2A-EGFP donor and sgAAVS1-mCherry expression vectors 24 h after dgRNA-Com:CK and/or dgRNA-MS2:MPH transfection. At day 3, the frequency of EGFP+ cells within mCherry+ population were determined using FACS. Data are showed as the mean ± SEM from three independent experiments. Significance was calculated using the Paired t-test. *p < 0.05, **p < 0.01, ***p < 0.001

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