Fig. 1

The schematic of CRISPR HITI. a In this strategy, two donor vectors without homology arms were utilized. These donner vectors contain sgRNA + PAM and EGFP expression cassette. In the guide of two sgRNAs, the Cas9 protein towards the exons 4 and 5 of CASP-7 at both alleles. Endonuclease function of Cas9 results in the deletion of a genomic fragment (between the exon 4 and 5) from both alleles, and the linearization of two donor vectors. The selection of cells for green color leads to the generation of caspase 7 knocked out cells. b The presentation of the HITI method, which uses NHEJ-mediated targeted integration in combination with Cas9 nuclease activity. If the target site remains intact or with integration in the reverse direction, DNA bears additional cleavage since forward gene insertion or gRNA can no longer bind to the target site through errors from the NHEJ repair system. c The schematic of PX460-1 vector which contains a single cloning site for bait gRNA and the expression cassette of the EGFP gene