Abstract：Nondret002679 gene is a type of long non-coding RNA (lncRNA) in zebrafish (Danio rerio) homologous to the LNC-PHOX2B-2 gene of large intergenic noncoding RNA 682 (lincRNA) in human (Homo sapiens), which is related to tumor and might be silenced by deleting the nucleotide sequences within its promoter region through clustered regularly interspaced short palindromic repeats/CRISPR- associated protein 9 (CRISPR/Cas9) system, to facilitate its function research. In the present study, the sequence positioned upstream and downstream region of its predicted promoter were used to design guide RNA (gRNA) to target its promoter region. There were 6 gRNAs located in either upstream or downstream promoter region, named as gR1, gR2, gR3, gR4, gR5 and gR6, respectively, were designed. These gRNAs were synthesized by in vitro transcription with the templates, which were made with 3 oligonucleotides by overlap PCR, a rapid method to obtain sufficient templates for gRNA. The 6 gRNAs and Cas9 mRNA were transcribed using transcription Kit and purified by LiCl precipitation and redissolved in RNase-free water. To knock out the sequences located in the predicted promoter of the lncRNA gene, a mixture of these gRNAs and Cas9 mRNA were microinjected into each zebrafish embryo at the one-cell stage with 1.76 nL. The concentration of each gRNA in injection solution was approximately 12.5 ng/μL, which also contained 300 ng/μL of Cas9 mRNA and 0.5% phenol red. Regular PCR with sequence-specific primers were used to identify individual knockout zebrafish with a deletion in the promoter region, showing a 3 643 bp PCR product in wild type zebrafish and 579~1 664 bp PCR products range in promoter deleted zebrafish. Twenty eight 2-month-old injected zebrafishes were extracted genomic DNA of their fin for PCR, out of which 11 were identified 579~1 664 bp PCR fragments range. Sequencing results, when compared to NCBI databases, demonstrated that 11 zebrafishes occurred deletion in lncRNA promoter region with 39% of knockout efficiency. The length of deletion fragments in 11 zebrafishes ranged of 1.9~3.1 kb. In addition, 33 F1 zebrafishes obtained from the breeding of one female and one male adult founders with heterozygous deletion were screened by PCR for deletion in the promoter region. The result indicated that the 6 F1 zebrafishes had promoter deletion with fragment range of 579~1 664 bp, which occurred homozygous deletion in lncRNA promoter in 3 zebrafishes. The result indicated that CRISPR/Cas9 system was an efficient approach to delete lncRNA gene promoter, which provides basic data for function study of Nondret002679 gene.
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