HITI is a CRISPR/Cas9-based strategy that enables gene insertion in non-dividing cells in vivo and in vitro and has applications in gene therapy.
HITI uses an NHEJ-based homology-independent strategy. Research lead by at the Salk Institute for Biological Studies in , suggests that error-free repair is dominant in their CRISPR/Cas9-based HITI method and that the method is more efficient than HDR. Guide RNAs (gRNAs) were used to specify the direction of the insertion. Classical HDR-based CRISPR/Cas9 genome-editing involves transfecting cells with , gRNA and donor DNA containing homologous arms matching the genomic locus of interest. For HITI, donor plasmids lack homology arms and DSB does not occur through the HDR pathway. The donor DNA includes Cas9 cleavage site(s) flanking the donor sequence resulting in Cas9 cleavage at both the donor plasmid and the genomic target sequence. Both target and donor have blunt ends and the linearized donor DNA plasmid is used by the NHEJ pathway resulting integration into the genomic DSB site. When incorporated in the correct orientation, the Cas9 target sequence is disrupted preventing further Cas9 cutting.