Fig. 5.

Two-cut MITI and negative selection strategy using Cas12a improved the accuracy of both junctions. a Schematics of the donor plasmid and targeting strategy for CRISPR/Cas12a-mediated insertion of the SA-IRES-GFP reporter at the AAVS1 locus. The donor plasmid carries two modified Cas12a target sites at both the 5′ and 3′ sides of SA-IRES-GFP (two-MITI donor, D7) which correspond to the genomic target sites, and a negative selection gene herpes simplex virus thymidine kinase (HSV-tk) in the prokaryotic backbone of the donor. b Detection of targeted integration of SA-IRES-GFP cassette from puromycin and FIAU dual resistant clones via PCR. Seven out of eleven clones had expected integration at the left and right junctions. c The sequencing results of the 5′- and 3′-integration junctions amplified from GFP-positive clones produced by the two-MITI donor