%0 Generic %A Li, Minghui %A Liu, Xingyong %A Dai, Shengfei %A Xiao, Hehsneg %A Wang, Deshou %D 2018 %T Supplemental Material for Li et al., 2018 %U https://gsajournals.figshare.com/articles/dataset/Supplemental_Material_for_Li_et_al_2018/7331054 %R 10.25387/g3.7331054.v1 %2 https://gsajournals.figshare.com/ndownloader/files/13541282 %K CRISPR/Cas9 %K non-coding sequence %K ssDNA %K germline transmission %K Biological Techniques %K Biotechnology %K Molecular Biology %X

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' 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-vasa 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 vasa gene was successfully deleted by dual gRNAs. Deletion of vasa 3'-UTR resulted in low expression level of vasa 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.

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