Targeted gene editing of an infectious salmon anemia (ISA)-relevant gene in salmonids cells using the CRISPR/Cas9 Ribonucleoprotein complex

Abstract

Background: The gene editing tool, CRISPR/Cas9, can potentially be applied in aquaculture to combat some of the problems of the farmed salmon industry including infectious diseases. The RNP (ribonucleoprotein)-based CRISPR/Cas9 strategy has recently been shown to be efficient in gene editing in diverse cell lines and organisms. Application of RNP strategy in salmon cell biology is still at its infancy. In this study, an in-house synthesized sgRNA (single guide ribonucleic acid) complexed to a Cas9-EGFP (EGFP; enhanced green fluorescent protein) was used to edit the cr2 gene (complement receptor 2) which is an immune gene regulated during infection of Atlantic salmon (Salmo salar) by infectious salmon anemia virus. The target cells in this study were the salmonid ASK-1 and CHSE-214 cell lines. Strategies for isolation and expansion of edited single cell clones were also established.

Methods: Site specificity and cleavage potentials on the cr2 gene of three sgRNAs (single guide ribonucleic acid) complexed to Cas9 were assessed using the in vitro cleavage assay prior to cell transfection of the complex. Transfection of the selected sgRNA/Cas9-EGFP complex was achieved by electroporation, and evaluation of in vivo activity was done using the T7 endonuclease 1 (T7E1) mismatch detection assay. Flow cytometry assisted cell sorting (FACS) enabled enrichment of the electroporated cells prior to detection of mutation by a combination of Sanger sequencing and the bioinformatic tools ICE and DECODR.

Results: The sgRNAs/Cas9 complex showed targeted in vitro cleavage activity of the cr2 gene, which was supported by the in vivo T7E1 mismatch assay following transfection in ASK-1 and CHSE-214 cells. A high electroporation rate of the RNP complex (approx. 85%) was achieved. Gene edits were successfully detected in the genome of the edited cells although with variable editing efficiency (100% and 5%) due to the heterogenous nature of the FACS enriched cell population. Isolation and expansion of edited single cell clones was achieved but could not be reproduced within the timeframe of this project.

Conclusion: This project successfully applied the CRISPR/Cas9 RNP complex strategy to mutate the cr2 gene in ASK-1 and CHSE-214 cells. The partial success in isolation and expansion of single clones of edited cells, and the identification of pitfalls as well as future perspectives related to the use of RNP complex in salmonids cells will contribute to extend this new frontier.