Abstract

CRISPR/Cas9 system has been used widely in animal and plants as an efficient genome editing tool, but it is no commonly used in salmonids, since it is not easy to implement due to the lack of fish specific promoters for the expression of sgRNAs and Cas9. Here we show the development of a CRISPR/Cas9 lentiviral expression system for fish. For this purpose we analyze the efficacy of different promoters in different fish cell lines in order to assess the expression of Cas9. We constructed several expression plasmids encoding the protein spCas9 driven by CMV and/or Ubiquitin (mammalian) promoter in a bicistronic cassette using mCherry as a reporter gene. For the expression of the sgRNA cassette we have chosen the U6 zebrafish promoter (Shinya et al. 2003). The expression of the Cas9/mCherry was assessed by DNA transfection using Lipofectamine 3000, which showed to be more efficient that CalfectinTM and TransIT for expression of the FUGpuroW plasmid in CHSE-214 and SHK-1 cell lines. The cell line derived from a Chinook salmon embryo (CHSE-214) and Lipofectamine 3000 exhibited the best combination for a high transfection efficiency at 96 hours post transfection and these conditions were used thereafter. As reported previously, the CMV promoter is able to drive the gene expression in fish cells, allowing to obtain stable fish line cell (CHSE-214) that overexpress a cytosolic chimeric protein eGFP:Puromycin. Interestingly, the mammalian ubiquitin promoter was also able to induce the expression of the eGFP, although with a lower fluorescence intensity suggesting that the CMV promoter is an appropriate promoter to drive the gene expression in this fish line cell. This is the first approach for developing a genome editing system in salmonids using lentiviral vectors, which could result in a powerful biotechnological tool for the salmon industry.