Efficient enrichment of genome-targeted cells using a surrogate reporter. (A) Enrichment of genome-targeted cells using a surrogate reporter (Puro). HeLa cells were cotransfected with Cas9, sgRNAs, a surrogate reporter (Puro), GFP, and PBase plasmids. Transfection-positive cells (GFP+) were divided into two groups: One group was treated with a high dose (2 µg/mL) of puromycin for 5 days, and the other group remained untreated. Targeting efficiency of these two group cells was determined by ICE analysis from Sanger sequencing. Cell colonies were picked for further genotyping. (**) P ≤ 0.01, (***) P ≤ 0.001 (Student's two-tailed t-test). (B) Enrichment of CMAH- and GGTA1-targeted PFFs by surrogate reporter (Neo) and surrogate reporter (GFP), respectively. (C) Enrichment of Tet2-targeted mESCs by surrogate reporter (Neo) and of et1-, Tet2-, and Tet3-targeted mESCs by surrogate reporter (eGFP), respectively. (D) The GFP expression in genomic targeted and nontargeted mESCs clones selected by the surrogate reporter (eGFP) plasmid. Scale bars, 1000 μm.
