Fig. 2

The LentiFlash® system can be used for the efficient CRISPR/Cas9-mediated modification of hiPSC. a Fluorescent microscopy analysis of HY03-GFP cells at day 6 post-transduction with LF-CRISPR/Cas9-GFP particles at 0.5, 2, or 5 pg p24/cell. Wild-type cells (WT) are parental HY03 cells. b Transduction efficacy measured by flow cytometry quantification of GFP-positive HY03-GFP cells at day 6 post-transduction with LF-CRISPR/Cas9-GFP particles at 0.5, 2, or 5 pg p24/cell (NT, not transduced = 100%). c Indel rate at five loci (GFP, MCIDAS, DNAH5, TRAC, and CXCR4) using increasing doses of LentiFlash® particles harboring CRISPR/Cas9 to target the indicated genes (from 0.1 to 10 pg p24/cell) in HY03 hiPSC cells at day 3 post-transduction. Data were obtained by ICE decomposition analysis after Sanger sequencing of the targeted loci. d Fold change of Cas9 mRNA level in HY03 cells transduced with LF-CRISPR/Cas9-MCIDAS or LF-CRISPR/Cas9-CXCR4 (0.5 and 7.5 pg p24/cell, respectively). The samples corresponding to cells analyzed at 24 h were arbitrarily defined as the reference (=1) (n = 1) e HRMA results of clones obtained following HY03 cells transduction with LF-CRISPR/Cas9-DNAH5 and LF-CRISPR/Cas9-MCIDAS particles at 0.5 pg p24/cell. WT, wild type. f Sanger sequencing analysis of clones identified as mutated by HRMA. Het, heterozygote; Comp-Het, compound heterozygote; Hom, homozygote