In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni

TLDR

CRISPR‑Cas9 orthologues have been employed for genome editing across diverse organisms. The study introduces the smallest Cas9 orthologue, CjCas9 from Campylobacter jejuni, aimed at efficient in vivo genome editing. CjCas9, together with optimized sgRNA and a marker gene, was packaged into a single‑vector adeno‑associated virus that was produced at high titer after PAM determination and sgRNA length optimization. CjCas9 displayed high specificity, induced high‑frequency targeted mutations in mouse muscle and retinal pigment epithelium cells, and reduced laser‑induced choroidal neovascularization when targeting Vegfa or Hif1a, indicating potential for treating age‑related macular degeneration.

Abstract

Abstract Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived from Campylobacter jejuni (CjCas9), for efficient genome editing in vivo . After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to the Vegfa or Hif1a gene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting that in vivo genome editing with CjCas9 is a new option for the treatment of age-related macular degeneration.