title: In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration.
abstract: Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Keiichiro
authors.initials: K
authors.lastname: Suzuki
authors.affiliation: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors.forename: Yuji
authors.initials: Y
authors.lastname: Tsunekawa
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.|4700 King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia.
authors.forename: Reyna
authors.initials: R
authors.lastname: Hernandez-Benitez
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.|Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain.
authors.forename: Jun
authors.initials: J
authors.lastname: Wu
authors.affiliation: Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China.|Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhu
authors.affiliation: Systems Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA.
authors.forename: Euiseok J
authors.initials: EJ
authors.lastname: Kim
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Fumiyuki
authors.initials: F
authors.lastname: Hatanaka
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Mako
authors.initials: M
authors.lastname: Yamamoto
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.|Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain.
authors.forename: Toshikazu
authors.initials: T
authors.lastname: Araoka
authors.affiliation: Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA.
authors.forename: Zhe
authors.initials: Z
authors.lastname: Li
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Masakazu
authors.initials: M
authors.lastname: Kurita
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Tomoaki
authors.initials: T
authors.lastname: Hishida
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Mo
authors.initials: M
authors.lastname: Li
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Emi
authors.initials: E
authors.lastname: Aizawa
authors.affiliation: Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA.
authors.forename: Shicheng
authors.initials: S
authors.lastname: Guo
authors.affiliation: Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA.
authors.forename: Song
authors.initials: S
authors.lastname: Chen
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: April
authors.initials: A
authors.lastname: Goebl
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Rupa Devi
authors.initials: RD
authors.lastname: Soligalla
authors.affiliation: State Key Laboratory of Stem Cell and Reproductive Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China.|University of Chinese Academy of Sciences, Beijing 100049, China.
authors.forename: Jing
authors.initials: J
authors.lastname: Qu
authors.affiliation: Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA.|Guangzhou EliteHealth Biological Pharmaceutical Technology Company Ltd, Guangzhou 510005, China.
authors.forename: Tingshuai
authors.initials: T
authors.lastname: Jiang
authors.affiliation: Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China.|Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA.
authors.forename: Xin
authors.initials: X
authors.lastname: Fu
authors.affiliation: Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA.
authors.forename: Maryam
authors.initials: M
authors.lastname: Jafari
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Concepcion Rodriguez
authors.initials: CR
authors.lastname: Esteban
authors.affiliation: Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: W Travis
authors.initials: WT
authors.lastname: Berggren
authors.affiliation: Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain.
authors.forename: Jeronimo
authors.initials: J
authors.lastname: Lajara
authors.affiliation: Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain.
authors.forename: Estrella
authors.initials: E
authors.lastname: Nuñez-Delicado
authors.affiliation: Universidad Católica San Antonio de Murcia (UCAM) Campus de los Jerónimos, no. 135 Guadalupe 30107, Murcia, Spain.|Fundación Dr. Pedro Guillen, Investigación Biomedica de Clinica CEMTRO, Avenida Ventisquero de la Condesa, 42, 28035 Madrid, Spain.
authors.forename: Pedro
authors.initials: P
authors.lastname: Guillen
authors.affiliation: Hospital Clinic, University of Barcelona, IDIBAPS, 08036 Barcelona, Spain.
authors.forename: Josep M
authors.initials: JM
authors.lastname: Campistol
authors.affiliation: Laboratory for Cell Asymmetry, RIKEN Center for Developmental Biology, 2-2-3 Minatojima-Minamimachi, Chuo-ku, Kobe 650-0047, Japan.
authors.forename: Fumio
authors.initials: F
authors.lastname: Matsuzaki
authors.affiliation: University of Chinese Academy of Sciences, Beijing 100049, China.|National Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.|Key Laboratory of Regenerative Medicine of Ministry of Education, Institute of Aging and Regenerative Medicine, Jinan University, Guangzhou 510632, China.|Beijing Institute for Brain Disorders, Beijing 100069, China.
authors.forename: Guang-Hui
authors.initials: GH
authors.lastname: Liu
authors.affiliation: 4700 King Abdullah University of Science and Technology (KAUST) Thuwal 23955-6900, Saudi Arabia.
authors.forename: Pierre
authors.initials: P
authors.lastname: Magistretti
authors.affiliation: Bioengineering, University of California, San Diego, 9500 Gilman Drive, MC0412, La Jolla, California 92093-0412, USA.
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Systems Neurobiology Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd., La Jolla, California 92037, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Callaway
authors.affiliation: Guangzhou Women and Children's Medical Center, Guangzhou Medical University, Guangzhou 510623, China.|Shiley Eye Institute, Institute for Genomic Medicine, Institute of Engineering in Medicine, University of California, San Diego, 9500 Gilman Drive #0946, La Jolla, California 92023, USA.|Molecular Medicine Research Center, West China Hospital, Sichuan University, Chengdu 610041, China.|Veterans Administration Healthcare System, San Diego, California 92093, USA.
authors.forename: Kang
authors.initials: K
authors.lastname: Zhang
authors.affiliation: Gene Expression Laboratory, Salk Institute for Biological Studies, 10010 N. Torrey Pines Rd, La Jolla, California 92037, USA.
authors.forename: Juan Carlos Izpisua
authors.initials: JC
authors.lastname: Belmonte
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002455:Cell Division; D004195:Disease Models, Animal; D000072669:Gene Editing / Q000379:methods*; D055879:Gene Knock-In Techniques; D018390:Gene Targeting / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods; D016678:Genome / Q000235:genetics*; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D051381:Rats; D012174:Retinitis Pigmentosa / Q000235:genetics* / Q000628:therapy*; D017385:Sequence Homology