Efficient tools that dissect gene function and enable introduction of desired genetic modifications at precise locations will radically advance existing genome improvement strategies in animal agriculture, especially in aquaculture. CRISPR/Cas9 has proven very effective to create knock-out alleles in multiple fish species via the non-homologous end-joining (NHEJ) pathway. However, precise insertion of exogenous donor DNA and gene swapping via the homology-directed repair (HDR) pathway has hardly ever been described in aquaculture species. We have achieved high frequency of precise knock-in of donor DNA and further demonstrated the possibility to replace and repair a deficient allele at equally high efficiency in tilapia.
Our strategy co-targeted pigment genes and genes affecting sterility (multiplexing) and used pigment defect as visual selection criteria to identify individuals more likely to carry the desired change .
We successfully generated tilapia lines where b -globin 3’UTR was integrated downstream of dead-end1 (dnd1) coding sequence. We measured ~ 50% of precise homology-directed knock-in amongst depigmented larvae following multiplex gene modification. F2 tilapia homozygous for the b-globin 3’UTR integration developed into sterile adults with un-developed ovaries and testes, revealing the essential role of dnd1-3’UTR in the maintenance of adult germ cells.
In addition to the homology directed knock-in, we successfully repaired a mutant version of the tyrosinase pigment gene for a wild-type version. An albino line of tilapia carrying a 7-nucleotide deletion at the tyrosinase locus (Tyralb7) was used. We observed the return of pigmented melanophores in 8% of injected embryos and showed germ line transmission of the corrected allele at frequency between 10% and 50%.
Our study indicates that precise genomic modification can be achieved by HDR in tilapia at high efficiency. These results open exciting possibilities in breeding programs allowing, for example, rapid introgression of favorable or de novo alleles into a breeding population opening new possibilities to improve health, welfare and performance of farmed fish by increasing disease resistance, growth rate or enhancing flesh quality and taste.