利用CRISPR/Cas9系统高效敲除斑马鱼lncRNA基因启动子区  

Efficient Knockout of lnc RNA Promoter Region by CRISPR/Cas9 System in Zebrafish(Danio rerio)

作　　者：田净净[1,2] 刘洋洋 杨哲[1,2] 刘菲菲[2] 杨祎擎 韩笑[1,2] 耿拓宇[1,2] 高波[1,2] 宋成义[1,2] 崔恒宓[1,2] 

机构地区：[1]扬州大学表观遗传学与表观基因组学研究所,扬州225009 [2]扬州大学动物科学与技术学院,扬州225009

出　　处：《农业生物技术学报》2016年第5期649-656,共8页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.81171965;No.81372237和No.91540117)

摘　　要：斑马鱼(Danio rerio)长非编码RNA(long non-coding RNA,lnc RNA)基因Nondret002679与人(Homo sapiens)肿瘤相关基因间长非编码RNA682基因LNC-PHOX2B-2具有同源序列。本研究以斑马鱼为模型,利用成簇规律间隔短回文重复序列/成簇规律间隔短回文重复序列关联蛋白9(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9,CRISPR/Cas9)系统,敲除预测的Nondret002679基因启动子区以沉默Nondret002679基因的表达,研究lnc RNA基因Nondret002679在斑马鱼中的调控功能。首先利用启动子区上下游序列设计靶向不同位点的向导RNA(guide RNA)g R1、g R2、g R3、g R4、g R5和g R6。人工合成g RNA转录模板,体外转录为g RNA,与Cas9 m RNA一起显微注射到斑马鱼卵中。PCR鉴定28尾2月龄斑马鱼发现,11尾发生敲除,敲除率为39%。繁殖启动子区敲除的杂合鱼,获得33尾F1代,经鉴定有6尾F1斑马鱼启动子区缺失,其中3尾缺失是在两条同源染色体上。该初步研究表明,CRISPR/Cas9系统可高效敲除lnc RNA基因的启动子区,并且这种敲除可以遗传至下一代,为研究lnc RNA功能提供了一个有效的基因编辑工具。NondretO02679 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 toobtain sufficient templates for gRNA. The 6 gRNAs and Cas9 mRNA were transcribed using transcription Kit and purified by LiC1 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/lzL, which also contained 300 ng/IzL 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 IncRNA promoter region with 39% of knocko

关 键 词：CRISPR/Cas9 向导RNA(gRNA) 长非编码RNA(lncRNA) 显微注射 Nondret002679 斑马鱼 

分 类 号：Q812[生物学—生物工程]