Figure 2
From: Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins
Increase in knock-in efficiency in HEK293T cells and enhanced multiplex indel efficiency of chimeric proteins in iPSCs. (A) Schematic of a 106-mer single-stranded oligonucleotide (ssODN). Artificially introduced NdeI restriction site (blue triangle) was flanked with homologous 5′ arm (57-mer) and 3′ arm (43-mer). (B) Comparison of homology-directed DNA repair (HDR) activity among C9, C9R, and C9G. HDR efficiency was evaluated by targeted deep sequencing of the CCR5, HPRT1, and EMX1 loci (left to right) after genome editing with the specific 106-mer template shown in panel a. (****P < 0.0001 and ***P < 0.001, one-way ANOVA using Tukey’s multiple-comparison test). For each gene, fold change was calculated relative to C9 efficiency. (C) Four different RNPs, each preassembled with a different sgRNA (targeting B2M, CIITA, CTLA4, or PDCD-1) were simultaneously transfected into induced pluripotent stem cells (iPSCs) and incubated for 24 h, and the targeted deep-sequencing analysis was performed to determine the indel efficiencies (****P < 0.0001, ***P < 0.001, and **P < 0.01, two-way ANOVA using Tukey’s multiple-comparison test).
