Digenome for off-target analysis: Start codon disruption with CRISPR/Cas9 prevents murine Fuchs' endothelial corneal dystrophy

A missense mutation of collagen type VIII alpha 2 chain (COL8A2) gene leads to early onset Fuchs' endothelial corneal dystrophy (FECD), which can cause blindness through progressive loss of corneal endothelial cells. We established a novel procedure for achieving structural and functional rescue of the post-mitotic corneal endothelium without surgery, using CRISPR/Cas9-based postnatal gene editing in a mouse model of FECD. A single intraocular injection of an adenovirus encoding both the Cas9 gene and guide RNA (Ad-Cas9-Col8a2gRNA) at a titer below the cytotoxic threshold, efficiently knocked down COL8A2 protein expression in corneal endothelial cells, prevented endothelial cell loss, and rescued corneal endothelium pumping function in adult Col8a2 mutant mice. Here, we evaluated the off-target activity of humanized gRNA targeting COL8A2 gene by a modified digenome analysis (Nat Methods 12, 237-243). Briefly, in vitro SpCas9 digested genomic DNA with/without gRNA are compared by deep sequencing. The target sequence is CGTCCACGGACGCCATGCT. In this study, we identified 13 candidate sites with homology to the gRNA and 8 candidate sites without homology to the gRNA. Overall design: Deep sequencing comparison between genomic DNAs which were digested by SpCas9 with/without gRNA.