Fig. 1: Schematic of the AAV-HITI therapeutic approach.
From: Therapeutic homology-independent targeted integration in retina and liver
AAV-delivered SpCas9 targets both the endogenous locus of a targeted gene and the inverted gRNA target sites flanking the donor DNA. Non-homologous end joining (NHEJ) recruits the donor DNA at the targeted locus. STOP codons knockout the endogenous gene while START sites initiate translation of the therapeutic gene. AAV adeno-associated viral vector, SpCas9 Streptococcus pyogenes Cas9, ITR inverted terminal repeats, U6-gRNA gRNA expression cassette containing the U6 promoter, the gene-specific or scramble gRNA and the gRNA scaffold; black rectangles and white triangles represent the two parts of the SpCas9 target sites flanking the SpCas9-induced double-strand break; scissors represent SpCas9-mediated double-strand breaks; STOP Stop codons in the 3 open reading frames, START translation start site (either kozak or internal ribosomal entry site, IRES), pA bovine growth hormone polyadenylation sequence; crossed scissors represent inability of SpCas9 to recognize target sites after homology-independent targeted integration has occurred.
