Supplemental Material for Li et al., 2018

<p>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 process. 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'<b> </b>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-<i>vasa</i> 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 <i>vasa</i> gene was successfully deleted by dual gRNAs. Deletion of <i>vasa</i> 3'-UTR resulted in low expression level of <i>vasa</i> 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.</p>