Fig. 4.

Tagging the CLTA gene using the two-cut donor in HepG2 cells. a Schematics of the donor plasmid and targeting strategy for CRISPR/Cas12a-mediated insertion of the 3 × FLAG-2A-tdTomato reporter at the CLTA locus in HepG2 cells. The two-cut donor (D4.1) includes two Cas12a recognition sites, one is the MITI-modified CrRNA target sequence, and the other is identical to the genomic target. The genetically modified CLTA locus may result in two different situations. One is that only the reporter part is knocked into the target site, and the other one is that the entire vector containing the prokaryotic backbone is integrated into the target site. b PCR identification of tdTomato-positive HepG2 clones. c Identification of tdTomato-positive HepG2 picked clones bearing predicted integration of 3 × FLAG-tdTomato by PCR and Sanger sequencing at the 5′ junction. d The sequences of the 3′ junction site of the representative HepG2 clone integrated with the whole D4.1 vector