Figure 5

In vivo genome editing via HMEJ-mediated targeted integration. (A) Experimental scheme for targeted Actb-2A-mCherry knock-in in fetal brain via in utero electroporation. (B) Representative immunofluorescence images of neurons showing correct mCherry knock-in at the Actb locus with four gene targeting strategies. Scale bar, 100 μm. GFP, transfected cells. (C) Relative knock-in efficiency measured by the percentage of mCherry⁺ cells among GFP⁺ cells. (D) Experimental scheme for targeted Actb-2A-mCherry knock-in via hydrodynamic tail vein injection. (E) Representative immunofluorescence images of hepatocytes in liver sections at day 7 post injection. Scale bar, 50 μm. GFP, transfected cells. (F) Relative knock-in efficiency measured by the percentage of mCherry⁺ cells among GFP⁺ cells. Hepatocytes were harvested at day 7 post injection. C and F, results were obtained from at least three mice and presented as mean ± SD. The input data points were shown as black dots. ^***P< 0.001, unpaired Student's t-test. (G) Schematic of HMEJ-AAV vectors for knock-in of p2A-mCherry to the last codon of the Actb gene. (H) Schematic of in vivo HMEJ-mediated knock-in via local AAV injections in adult mouse brain. (I) Representative immunofluorescence images of neurons in HMEJ-AAV-injected brain sections. Insets, higher magnification images. Scale bar, 100 μm. (J) Relative and absolute knock-in efficiencies measured by the percentage of mCherry⁺ cells among GFP⁺ cells or all DAPI⁺ cells, respectively. Results were obtained from two animals and presented as mean ± SD. At least 2 000 cells of each brain section and three brain sections of each animal were counted. The input data points were shown as black dots.