title: In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni.
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.
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.|ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea.
authors.forename: Eunji
authors.initials: E
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.|Department of Functional Genomics, University of Science and Technology, Daejeon 34113, Republic of Korea.
authors.forename: Taeyoung
authors.initials: T
authors.lastname: Koo
authors.affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.|FARB Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Republic of Korea.
authors.forename: Sung Wook
authors.identifier: 0000-0001-8151-6663
authors.initials: SW
authors.lastname: Park
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Daesik
authors.initials: D
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.
authors.forename: Kyoungmi
authors.initials: K
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.
authors.forename: Hee-Yeon
authors.initials: HY
authors.lastname: Cho
authors.affiliation: ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea.
authors.forename: Dong Woo
authors.initials: DW
authors.lastname: Song
authors.affiliation: ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea.
authors.forename: Kyu Jun
authors.initials: KJ
authors.lastname: Lee
authors.affiliation: ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea.
authors.forename: Min Hee
authors.initials: MH
authors.lastname: Jung
authors.affiliation: ToolGen, Byucksan Digital Valley 6-cha, 219 Gasan Digital 1-ro, Geumcheon-gu, Seoul 08501, Republic of Korea.
authors.forename: Seokjoong
authors.initials: S
authors.lastname: Kim
authors.affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.|FARB Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Republic of Korea.
authors.forename: Jin Hyoung
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.|FARB Laboratory, Biomedical Research Institute, Seoul National University Hospital, Seoul 03082, Republic of Korea.|Department of Ophthalmology, Seoul National University College of Medicine, Seoul 03080, Republic of Korea.
authors.forename: Jeong Hun
authors.initials: JH
authors.lastname: Kim
authors.affiliation: Center for Genome Engineering, Institute for Basic Science (IBS), Seoul 08826, Republic of Korea.|Department of Functional Genomics, University of Science and Technology, Daejeon 34113, Republic of Korea.|Department of Chemistry, Seoul National University, Seoul 08826, Republic of Korea.
authors.forename: Jin-Soo
authors.initials: JS
authors.lastname: Kim
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D016123:Campylobacter jejuni / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D020256:Choroidal Neovascularization / Q000235:genetics; D000229:Dependovirus / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing / Q000379:methods*; D057809:HEK293 Cells; D006801:Humans; D008297:Male; D008810:Mice, Inbred C57BL; D055213:Retinal Pigment Epithelium / Q000166:cytology / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D042461:Vascular Endothelial Growth Factor A; D004720:Endonucleases