The Application Of CRISPR/Cas9 System In The Zebrafish Gene Editing

With the completion of the zebrafish (Danio rerio) genome sequencing, researchers found that there is high homology between zebrafish and human(Homo sapiens). The researchers constructed many zebrafish model of the human diseases, for exploring the disease mechanisms. The application of gene editing techniques promotes gene function researches in zebrafish. A new gene editing technofogy, CRISPR/Cas9 system is widely used in gene editing, because of its ease and efficientcy. This study aims at deleting the promoter of lncRNA gene and knocking dCas9 gene in the genomic loci of zebrafish via CRISPR/Cas9 system1、 The Nondret002679 is a long non-coding RNA (lncRNA) gene in zebrafish. We investigated the expression patterns of Nondret002679 in different stage (0 hpf,3 hpf,6 hpf,12 hpf,24 hpf and 48 hpf) of zebrafish embryos and in different tissues (brain, muscle, testis, fin, ovary liver and heart) of adult zebrafish using reverse transcription quantitative PCR (RT-qPCR). The results indicated that the expression of Nondret002679 was increased 456 times from a single cell embryos to 24 h zebrafish (p< 0.01), and was increased 1065 times from a single cell embryos to 48 h (p< 0.01). The expression of Nondret002679 was increased during in first 4 stages, however there is exist no difference (p> 0.05). The expression of Nondret002679 in brain was 248 times higher than in heart (p<0.01) and was no difference between in other tissues, which indicated tissue-specific expression.2、Nondret002679 gene was silenced by deleting the sequences of its predicted promoter region through CRISPR/Cas9 system. The sequences upstream and downstream region of its promoter were used to design 6 guide RNAs (gRNAs), named as gRl, gR2, gR3, gR4, gR5 and gR6 gRNA, respectively. And a mixture of these gRNAs and Cas9 mRNA were micro injected into zebrafish embryo at the one-cell stage to knock out the promoter. Regular PCR with sequence-specific primers was used to identify individual zebrafish. The result showed that 11 out of 28 two-month-old zebrafish had a deletion in the promoter region of the gene with 39% of knockout efficiency. In 33 F1 zebrafishes obtained from the breeding of heterozygous founders, 3 F1 zebrafishes showed heterozygous deletion and 3 showed homozygous deletion in promoter. In addition, Nondret002679 transcription level was analysed by RT-qPCR in Fo zebrafishs with heterozygous deletion indicating that the transcription levels were decreased in 4/5 zebrafishs.3、 The establishment of a dCas9 transgenic zebrafish model via CRISPR/Cas9 system. The 522 dCas9 donor and 1092 dCas9 donor vocter were designed, according to the length of homologous arm. The results indicated that 522 donor group success knock-in ratio was 25.6%, concluding 40% homozygous zebrafish.1092 donor group success knock-in ratio was 20.5%, concluding 11% homozygous zebrafish. Meanwhile the dCas9 was succeessfully knocked-in and was detected to express in the transgenic zebraishes by PCR and RT-PCR, respectively.In conclusion, the CRISPR/Cas9 system could effectively knockout the promoter region of lncRNA gene, leading to reduced expression of incRNA in zebrafishes with deletion. Therefore, the CRISPR/Cas9 system may provide an efficient gene editing tools for the study of IncRNA function. The zebrafish model expressing dCas9 was obtained by CRISPR/Cas9 system, which may be very useful for the study of gene function.