Efficient CRISPR/Cas9 genome editing in a salmonid fish cell line using a lentivirus delivery system

被引:39
|
作者
Gratacap, Remi L. [1 ]
Regan, Tim [1 ]
Dehler, Carola E. [2 ]
Martin, Samuel A. M. [2 ]
Boudinot, Pierre [3 ]
Collet, Bertrand [3 ]
Houston, Ross D. [1 ]
机构
[1] Univ Edinburgh, Roslin Inst, Easter Bush Campus, Edinburgh, Midlothian, Scotland
[2] Univ Aberdeen, Inst Biol & Environm Sci, Aberdeen, Scotland
[3] Univ Paris Saclay, Virol & Immunol Mol, Inst Natl Rech Agron INRA, Jouy En Josas, France
基金
英国生物技术与生命科学研究理事会;
关键词
CRISPR; Lentivirus; Gene editing; CHSE; Salmon; Disease resistance; VIRUS; VECTORS; TRANSDUCTION; FUTURE; GENES;
D O I
10.1186/s12896-020-00626-x
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
Background Genome editing is transforming bioscience research, but its application to non-model organisms, such as farmed animal species, requires optimisation. Salmonids are the most important aquaculture species by value, and improving genetic resistance to infectious disease is a major goal. However, use of genome editing to evaluate putative disease resistance genes in cell lines, and the use of genome-wide CRISPR screens is currently limited by a lack of available tools and techniques. Results In the current study, we developed an optimised protocol using lentivirus transduction for efficient integration of constructs into the genome of a Chinook salmon (Oncorhynchus tshwaytcha) cell line (CHSE-214). As proof-of-principle, two target genes were edited with high efficiency in an EGFP-Cas9 stable CHSE cell line; specifically, the exogenous, integrated EGFP and the endogenous RIG-I locus. Finally, the effective use of antibiotic selection to enrich the successfully edited targeted population was demonstrated. Conclusions The optimised lentiviral-mediated CRISPR method reported here increases possibilities for efficient genome editing in salmonid cells, in particular for future applications of genome-wide CRISPR screens for disease resistance.
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页数:9
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