Aquaculture America 2020

February 9 - 12, 2020

Honolulu, Hawaii

MAKING PRESCISE GENETIC CHANGES IN THE TILAPIA GENOME

Takeshi Umazume , Melissa Hoffman, Valerie Williams,  John Buchanan, Xavier Lauth
 The Center for Aquaculture Technologies
 8395 Camino Santa Fe, San Diego, CA
xlauth@aquatechcenter.com
 

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.