Saltar al contenido
Merck

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

BMC biotechnology (2020-06-25)
Remi L Gratacap, Tim Regan, Carola E Dehler, Samuel A M Martin, Pierre Boudinot, Bertrand Collet, Ross D Houston
RESUMEN

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. 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. 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.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
L-15 Medium (Leibovitz), With L-glutamine, liquid, sterile-filtered, suitable for cell culture