Fig. 1. HITI experimental design.

(A) HITI minicircles (MCs) contain a Cas9 cut site identical to that at the AAVS1 safe harbor locus. Both genomic and MC DNA are cut in the presence of a gRNA and Cas9. Genes of interest (GOI) are only stably integrated into the genome when inserted in the correct orientation; otherwise, the Cas9 cut sites are preserved, which increases the likelihood of continuous Cas9 cutting. (B) Trimodality HDR, HITI, and Cas9 MC constructs designed for this study. (C) Restriction digest agarose gel of parental plasmids (PPs) and MCs and indicated band sizes. (D) Transfection regimen for combinations of donor and Cas9 MCs and simplified abbreviations for each condition.