CRISPR-Cas9/Cas12a-based genome editing in Atlantic cod (Gadus morhua)

Aquaculture is the fastest-growing food sector worldwide but faces sustainability challenges that need to be addressed in many ways, including genetic enhancement. Atlantic cod has re-emerged as an aquaculture species and tools for genetic manipulation are needed. Thus, we compared five formats of CRISPR to determine which was most efficient to generate knock outs in Atlantic cod. Cas9 protein was presented in preformed ribonucleoprotein (RNP) complexes with single guide or with duplex guide RNAs or an mRNA encoding Cas9 was used with the same two formats of guide RNAs. Cas12a was tested as RNP complexes with single guide RNAs. We found Cas9 mRNA with single guide RNA to be the most efficient format to knock out both alleles of the slc45a2 gene, which resulted in an albino-like phenotype in up to 75% of surviving larvae. DNA analysis of individual larvae revealed mosaic genotypes with variable indel mutations. The mortality of injected eggs was high, resulting in low overall efficiency. Nevertheless, this study lays the foundation for further genetic and functional research using the CRISPR/Cas9 genome editing system in Atlantic cod.