Figure 1.

Delivery of CRISPR/Cas9 Mediates Gene Editing In Vitro

(A) Experimental design. “A” in green indicates the WT base. Two sgRNAs (underlined) were designed according to the mutation site of four bases (red in the MUT lines). PAM is indicated with yellow frame; blue vertical lines mark the cut sites. Heterozygous KI-Krt9 mice harboring the indel mutant, c.434delAinsGGCT (p.Tyr144delinsTrpLeu), treated with sg1-Cas9-LV after in vitro experiments confirmed the efficacy of the LV vector. (B and C) FACS results showing the changes in FITC and mCherry fluorescence after treating HeLa cells with LV expressing both Krt9 exon1-EGFP and Krt9 exon1 MUT-mCherry (B). Thus, the HeLa Hez cell line was established. The reduction of EGFP fluorescence after transduction by sg1-Cas9-LV or sg2-Cas9-LV is shown in (C). See also Figure S2. (D and E) Immunoblotting of HeLa Hez cells against K9 (D) and semiquantitative immunoblotting analyses (relative to GAPDH and FLAG expression) (n = 3, *p < 0.05) (E). Each experiment was repeated at least three times. NC, untreated HeLa Hez cells; PC, HeLa Hez cells treated with Cas9-LV.