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ABSTRACT Studies of gene function in non-model animals have been limited by the approaches available for eliminating gene function. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated) system has recently become a powerful tool for targeted genome editing. Here, we report the use of the CRISPR/Cas9 system to disrupt selected genes, including nanos2, nanos3, dmrt1, and foxl2, with efficiencies as high as 95%. In addition, mutations in dmrt1 and foxl2 induced by CRISPR/Cas9 were efficiently transmitted through the germline to F1. Obvious phenotypes were observed in the G0 generation after mutation of germ cell or somatic cell-specific genes. For example, loss of Nanos2 and Nanos3 in XY and XX fish resulted in germ cell-deficient gonads as demonstrated by GFP labeling and Vasa staining, respectively, while masculinization of somatic cells in both XY and XX gonads was demonstrated by Dmrt1 and Cyp11b2 immunohistochemistry and by up-regulation of serum androgen levels. Our data demonstrate that targeted, heritable gene editing can be achieved in tilapia, providing a convenient and effective approach for generating loss-of-function mutants. Furthermore, our study shows the utility of the CRISPR/Cas9 system for genetic engineering in non-model species like tilapia and potentially in many other teleost species.
RECENTLY, a simple and ef fi cient genome editing tech- nology, type II CRISPR/Cas9, has been developed based on the Streptococcus pyogenes clustered regularly inter- spaced short palindromic repeats (CRISPR)-associated pro- tein (Cas9) adaptive immune system. It requires three components for effective DNA cleavage: the nuclease Cas9, a targeting CRISPR RNA (crRNA), and an additional trans - activating crRNA (tracrRNA) (Gasiunas et al. 2012; Jinek et al. 2012; Cho et al. 2013; Cong et al. 2013; Hwang et al. 2013; Mali et al. 2013). Further improvement of the system was achieved by fusing the crRNA and tracrRNA to form a single guide RNA (gRNA) that is sufficient to direct Cas9-mediated target cleavage (Hwang et al. 2013). Impor- tantly, previous studies performed in vitro (Jinek et al. 2012), in bacteria (Jiang et al. 2013), and in human cells (Cong et al. 2013) have shown that Cas9-mediated cleavage can be abol- ished by single mismatch at the gRNA-target site interface, particularly in the last 10-12 nucleotides located in the 39 end of the 20-nt gRNA targeting region. Compared...