Fig. 2

CRISPR-Cas9-mediated gene knock-in using the homology-independent targeted integration (HITI) strategy. A The CRISPR-Cas9-mediated gene editing initiates after the delivery of CRISPR-Cas9 machinery into target cells. The HITI strategy consists of two steps, including CRISPR-Cas9-mediated DSB formation and DNA repair via the NHEJ pathway. B–D An example of RS1/GFP knock-in using the HITI strategy. B Representative bright-field and fluorescence images of RS1/GFP-knock-in B16 cells. C PCR analysis showing the presence of right-arm (R-arm) junction (617 bp) and left-arm (L-arm) junction (748 bp) after the integration of RS1/GFP into the ROSA26 sites. D Sanger sequencing of the genome-donor boundaries in the R-arm and L-arm junctions confirming the integration of RS1/GFP. E Quantitative PCR analysis showing the upregulation of RS1 gene after the RS1/GFP gene knock-in. F Immunofluorescence staining showing the expression of RS1 and GFP after the RS1/GFP gene knock-in. All data are reproduced from our previous work [119]