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. Recently the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated proteins (Cas) system has been adapted to serve as a targeted genome mutagenesis tool. CRISPR/Cas9 have recently been used to create knock-out alleles with great efficiency in multiple organisms, including fish. Here we report expansion of targeted genome modification repertoire in tilapia. Using CRISPR/Cas9 and circular donor DNA we achieved high frequency of precise knock-in of foreign sequence and further demonstrated the possibility to replace and repair a mutant allele at equally high efficiency .
Our strategy co-targeted pigment genes and used pigment defect as selection markers to identify individuals carrying the desired modification.
We successfully generated tilapia lines where β -globin 3'UTR was integrated downstream of dead-end1 (dnd1) coding sequence . We obtained close to 50% of larvae with precise homology-directed knock-in amongst selected embryos. F2 tilapia homozygous for β -globin 3'UTR integration developed into sterile adult with string-like ovaries and translucid testes, revealing the essential role of dnd1-3'UTR in the maintenance of adult germ cells.
In addition to the h omology directed knock-in, we attempted to swap a mutant version of the tyrosinase pigment gene for a wild type version. Here we used an albino line of tilapia carrying a 7-nucleotide deletion at the tyrosinase locus (Tyralb7). We show repair of Tyralb7 in as high as 8% of injected embryos, as visualized by the appearance of mosaic pigmented melanophores . We further found germ line transmission of the corrected allele in frequency between 10% andto 50%.
Our study indicates that precise genomic modification can be achieved by homology directed repair in the tilapia genome with high efficiency. These results open exciting possibilities for further improvement in breeding programs, allowing for example, rapid introgression of favorable or de novo alleles into a breeding population.