title: Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line.
abstract: CRISPR/Cas9 system has been used widely in animals and plants to direct mutagenesis. To date, no such method exists for fish somatic cell lines. We describe an efficient procedure for genome editing in the Chinook salmon Oncorhynchus tshawytscha CHSE. This cell line was genetically modified to firstly overexpress a monomeric form of EGFP (cell line CHSE-E Geneticin resistant) and additionally to overexpress nCas9n, a nuclear version of Cas9 (cell line CHSE-EC, Hygromycin and Geneticin resistant). A pre-validated sgRNA was produced in vitro and used to transfect CHSE-EC cells. The EGFP gene was disrupted in 34.6 % of cells, as estimated by FACS and microscopy. The targeted locus was characterised by PCR amplification, cloning and sequencing of PCR products; inactivation of the EGFP gene by deletions in the expected site was validated in 25 % of clones. This method opens perspectives for functional genomic studies compatible with high-throughput screening.
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen Tillydrone Avenue, Aberdeen, AB24 2TZ, UK.
authors.forename: Carola E
authors.initials: CE
authors.lastname: Dehler
authors.affiliation: VIM, INRA, Université Paris-Saclay, 78350, Jouy-en-Josas, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Boudinot
authors.affiliation: Institute of Biological and Environmental Sciences, University of Aberdeen Tillydrone Avenue, Aberdeen, AB24 2TZ, UK.
authors.forename: Samuel A M
authors.initials: SA
authors.lastname: Martin
authors.affiliation: Marine Laboratory, Marine Scotland, 375 Victoria road, Aberdeen, AB11 9DB, UK. Bertrand.Collet@gov.scot.
authors.forename: Bertrand
authors.initials: B
authors.lastname: Collet
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D002460:Cell Line; D002934:Cinnamates; D003001:Cloning, Molecular; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D000072669:Gene Editing / Q000379:methods*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D005822:Genetic Vectors / Q000737:chemistry / Q000378:metabolism; D005839:Gentamicins; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006921:Hygromycin B / Q000031:analogs & derivatives; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism; D012474:Salmon / Q000235:genetics*; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D002934:Cinnamates; D005839:Gentamicins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D006921:Hygromycin B; C026273:hygromycin A; C010680:antibiotic G 418; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CHSE-214; CRISPR/Cas9; EGFP; FACS; Genome editing; nCas9n