High Efficiency Targeting of Non-coding Sequences Using CRISPR/Cas9 System in Tilapia.

The CRISPR/Cas9 has been successfully applied for disruption of protein coding sequences in a variety of organisms. The majority of the animal genome is actually non-coding sequences, which are key regulators associated with various biological processes. In this study, to understand the biological significance of these sequences, we used one or dual gRNA guided Cas9 nuclease to achieve specific deletion of non-coding sequences including microRNA and 3' untranslated region (UTR) in tilapia, which is an important fish for studying sex determination and evolution. Co-injection of fertilized eggs with single gRNA targeting seed region of miRNA and Cas9 mRNA resulted in indel mutations. Further, co-injection of fertilized eggs with dual gRNAs and Cas9 mRNA led to the removal of the fragment between the two target loci, yielding maximum efficiency of 11%. This highest genomic deletion efficiency was further improved up to 19% using short ssDNA as a donor. The deletions can be transmitted through the germline to the next generation at average efficiency of 8.7%. Cas9- 3'-UTR was used to increase the efficiency of germline transmission of non-coding sequence deletion up to 14.9%. In addition, the 3'-UTR of the gene was successfully deleted by dual gRNAs. Deletion of 3'-UTR resulted in low expression level of mRNA in the gonad when compared with the control. To summarize, the improved CRISPR/Cas9 system provided a powerful platform that can assist to easily generate desirable non-coding sequences mutants in non-model fish tilapia to discovery their functions.