Fig. 1

Insertional disruption of GFP transgene via NHEJ-based knock-in. a Schematic for NHEJ-based homology-independent knock-in of ires-Td reporter at the GFP transgene in LO2-GFP cells. sgGFP-i and sgGFP-ii are two different sgRNAs targeting GFP coding sequence. Shown are GFP transgene integrated at GAPDH locus, before and after the knock-in of ires-Td reporter. b FACS plots obtained after cotransfection of ires-Td_donor/Cas9/sg-A with sgGFP-i or sgGFP-ii in LO2-GFP cells. GFP⁺ cells are gated to the right, and Td⁺ cells are gated to the top in each plot. The control without sgRNA to GFP is shown. c Fluorescence images showing the expression of GFP transgene as well as newly integrated tdTomato reporter. Nuclei were stained using Hoechst. Arrows indicate the cells that have acquired tdTomato expression but lost the GFP expression. Scale bars = 50 μm