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

Nat Commun. 2017 Feb 21;8:14500. doi: 10.1038/ncomms14500.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • CRISPR-Cas Systems*
  • Campylobacter jejuni / genetics
  • Campylobacter jejuni / metabolism*
  • Cells, Cultured
  • Choroidal Neovascularization / genetics
  • Dependovirus / genetics
  • Endonucleases / genetics
  • Endonucleases / metabolism*
  • Gene Editing / methods*
  • HEK293 Cells
  • Humans
  • Male
  • Mice, Inbred C57BL
  • Retinal Pigment Epithelium / cytology
  • Retinal Pigment Epithelium / metabolism
  • Vascular Endothelial Growth Factor A / genetics
  • Vascular Endothelial Growth Factor A / metabolism

Substances

  • Bacterial Proteins
  • Vascular Endothelial Growth Factor A
  • Endonucleases