Abstract Selective breeding programs in aquaculture are limited by the heritability of the trait and long generation time in most fish species. New breeding technology (NBT) using CRISPR/Cas9-induced homology directed repair (HDR) have the potential to expedite genetic improvement in aquaculture, but the method requires optimization. Here we show that asymmetrical oligonucleotide (ODN) donors induce highly efficient and precise edits in individual Atlantic salmon founder animals. We performed single nucleotide replacement (SNR) in dnd with up to 59.2% efficiency, and inserted FLAG elements into slc45a2 and dnd , with up to 36.7 % and 32.7% efficiency, respectively. We found HDR efficiency to be dependent on template concentration, but a trade-off with respect to toxicity was observed. Using this NBT in salmon we demonstrate that precise modification of the genome can be achieved in a single generation, allowing efficient introgression of favorable alleles and bypassing challenges associated with traditional selective breeding.

A refinement to gene editing in Atlantic salmon using asymmetrical oligonucleotide donors