Fig. 3

Comparing RGN-induced gene targeting based on standard and in trans paired nicking in human PSCs. a Quantification of genetically modified PSCs by flow cytometry. Cultures of iPSCs (A, B, and E) and ESCs (C and D) were exposed to AAVS1-specific cleaving Cas9:gRNA^S1 (standard) or nicking Cas9^D10A:gRNA^S1 (Nick²) complexes mixed with RGN-resistant or RGN-susceptible donor constructs, respectively, encoding either EGFP or Puro^R.T2A.EGFP. The frequencies of gene-modified PSCs were determined by flow cytometric quantification of EGFP⁺ and TRA-1-81⁺ dually labeled cells. b Representative flow cytometry dot plots corresponding to RGN-induced gene targeting experiments in PSCs. c Detection of gene-modified PSCs by colony-formation assays. ESCs (top) and iPSCs (bottom) were co-transfected with the indicated plasmids. After puromycin selection, alkaline phosphatase staining identified genetically modified PSC colonies. d RGN-induced gene targeting frequencies at AAVS1 in iPSCs. Junction PCR analyses of puromycin-resistant colonies from iPSC cultures initially co-transfected with pDonor.EP^S1 and pCas9.gRNA^S1 (standard) or with pDonor.EP^S1.TS and pCas9^D10A.gRNA^S1 (Nick²). The respective PCR screening data are presented in Supplementary Fig. 13. e Differentiation potential of gene-edited PSCs. ESC and iPSC lines were targeted at AAVS1 by in trans paired nicking. Cell types characteristic of ectoderm, endoderm, and mesoderm were identified by confocal immunofluorescence microscopy for TUBB3, AFP, and CD31, respectively. f Characterization of indel footprints in iPSCs subjected to standard vs. in trans paired nicking. Nucleotide sequencing of AAVS1 target alleles in randomly selected iPSC clones (n = 68) genetically modified by DSB-dependent and in trans paired nicking methodologies (Standard and Nick², respectively). Indel footprints were exclusively identified in iPSCs subjected to the standard gene targeting approach (15/28). The gRNA^S1 target site is indicated underneath the sequence reads. Open box PAM; vertical dashed line position of expected RGN-induced phosphodiester bond cleavage; Ctrl reference wild-type nucleotide sequence from unedited cells