Abstract
Reverse genetics approaches are critical for uncovering complex biological processes and genetic engineering. Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated (Cas) system (CRISPR/Cas9) has been widely used for generating mutants in a large number of species. The Nile tilapia (Oreochromis niloticus), a gonochoristic teleost with XX/XY sex determination system, is a good model for studying fish sex determination and reproduction. Here, we describe a detailed procedure for gene mutation in tilapia by CRISPR/Cas9. This protocol covers selection of target sites, in vitro RNA transcription, artificial insemination and microinjection of one cell stage embryos. We also provide details for detection of somatic and germline transmitted mutations, fast establishment of mutant lines and discuss some practical advices. This protocol will facilitate broader applications of CRISPR/Cas9 in studies of tilapia as well as other aquaculture fishes.
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