Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

Carola E. Dehler, Pierre Boudinot, Samuel A. M. Martin, Bertrand Collet (Corresponding Author)

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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.
Original languageEnglish
Pages (from-to)449-452
Number of pages4
JournalMarine Biotechnology
Volume18
Issue number4
Early online date28 May 2016
DOIs
Publication statusPublished - Aug 2016

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Clustered Regularly Interspaced Short Palindromic Repeats
genome
geneticin
cell lines
Cell Line
gene
Salmon
Oncorhynchus tshawytscha
fish
microscopy
clone
amplification
genomics
Polymerase Chain Reaction
animal
Gene Deletion
methodology
somatic cells
mutagenesis
Mutagenesis

Keywords

  • CRISPR/Cas9
  • CHSE-214
  • EGFP
  • FACS
  • genome editing
  • nCas9n

Cite this

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line. / Dehler, Carola E.; Boudinot, Pierre; Martin, Samuel A. M.; Collet, Bertrand (Corresponding Author).

In: Marine Biotechnology, Vol. 18, No. 4, 08.2016, p. 449-452.

Research output: Contribution to journalArticle

Dehler, Carola E. ; Boudinot, Pierre ; Martin, Samuel A. M. ; Collet, Bertrand. / Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line. In: Marine Biotechnology. 2016 ; Vol. 18, No. 4. pp. 449-452.
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