title: Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.
abstract: Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
authors.forename: Vemulawada
authors.initials: V
authors.lastname: Chakrapani
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
authors.forename: Swagat Kumar
authors.initials: SK
authors.lastname: Patra
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
authors.forename: Rudra Prasanna
authors.initials: RP
authors.lastname: Panda
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
authors.forename: Kiran Dashrath
authors.initials: KD
authors.lastname: Rasal
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India.
authors.forename: Pallipuram
authors.initials: P
authors.lastname: Jayasankar
authors.affiliation: Fish Genetics and Biotechnology Division, ICAR - Central Institute of Freshwater Aquaculture, Bhubaneswar 751 002, Odisha, India. Electronic address: Hirak.Barman@icar.gov.in.
authors.forename: Hirak Kumar
authors.initials: HK
authors.lastname: Barman
mesh_terms: D000663:Amphibians; D000818:Animals; D017756:Aquaculture; D001426:Bacterial Proteins / Q000235:genetics; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002347:Carps / Q000276:immunology*; D004720:Endonucleases / Q000235:genetics; D029941:Fish Proteins / Q000235:genetics / Q000378:metabolism*; D000072669:Gene Editing; D055785:Gene Knockdown Techniques / Q000379:methods*; D059765:Homologous Recombination; D007113:Immunity, Innate; D051193:Toll-Like Receptors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D029941:Fish Proteins; D051193:Toll-Like Receptors; D000076987:CRISPR-Associated Protein 9; C000600049:Cas9 protein, Francisella novicida; D004720:Endonucleases
keywords: CRISPR/Cas9; Carp TLR22; Gene editing; Homologous recombination; Microinjection