Eunji Kim1,2,*, Taeyoung Koo1,3,*, Sung Wook Park4,5,*, Daesik Kim1,6, Kyoungmi Kim1, Hee-Yeon Cho1, Dong Woo Song2, Kyu Jun Lee2, Min Hee Jung2, Seokjoong Kim2, Jin Hyoung Kim4,5, Jeong Hun Kim4,5,7 & Jin-Soo Kim1,3,6
1 Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea. 2 ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea. 3 Department of Functional Genomics, University of Science and Technology, Daejeon 34113, Republic of Korea. 4 Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea. 5 FARB Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Republic of Korea. 6 Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea. 7 Department of Ophthalmology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.
* These authors contributed equally to this work.
Correspondence to Jeong Hun Kim or Jin-Soo Kim
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.