Supplemental Material for Li et al., 2018
Minghui Li
Xingyong Liu
Shengfei Dai
Hehsneg Xiao
Deshou Wang
10.25387/g3.7331054.v1
https://gsajournals.figshare.com/articles/dataset/Supplemental_Material_for_Li_et_al_2018/7331054
<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>
2018-11-27 17:59:08
CRISPR/Cas9
non-coding sequence
ssDNA
germline transmission
Biological Techniques
Biotechnology
Molecular Biology