Targeted mutagenesis using CRISPR/Cas system in medaka.
Targeted mutagenesis using CRISPR/Cas system in medaka.
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DOI：
10.1242/bio.20148177
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发表时间：
2014-04-11
期刊：
影响因子：
2.4
通讯作者：
Kinoshita M
中科院分区：
文献类型：
--
作者：
Ansai S;Kinoshita M
基于规则间隔短回文重复序列（CRISPR）/CRISPR相关（Cas）系统的RNA引导的核酸内切酶（RGEN）最近已经成为用于靶向基因组编辑的简单且有效的工具。在这项研究中，我们成功地利用RGEN在青鳉，青鳉的靶向诱变。体细胞和可遗传突变在已经注射了由T7启动子转录的单向导RNA（sgRNA）和编码Cas9核酸酶的加帽RNA的胚胎中的DJ-1基因上的靶向基因组序列处高效诱导。针对靶基因组序列设计的不具有T7启动子所需的GG的5′端的sgRNA诱导靶向突变。这表明RGEN可以靶向与NGG前间区序列邻近基序（PAM）序列相邻的任何序列，其每8bp出现一次。 在RGEN注射的胚胎中诱导了2个基因组位点的脱靶改变，这些基因组位点在18-bp靶向序列中具有双错配。然而，我们还发现，脱靶效应可以通过较低剂量的sgRNA来降低。综上所述，我们的研究结果表明，CRISPR/Cas介导的RGEN可能是青鳉基因组编辑的有效和灵活的工具。
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system-based RNA-guided endonuclease (RGEN) has recently emerged as a simple and efficient tool for targeted genome editing. In this study, we showed successful targeted mutagenesis using RGENs in medaka, Oryzias latipes. Somatic and heritable mutations were induced with high efficiency at the targeted genomic sequence on the DJ-1 gene in embryos that had been injected with the single guide RNA (sgRNA) transcribed by a T7 promoter and capped RNA encoding a Cas9 nuclease. The sgRNAs that were designed for the target genomic sequences without the 5′ end of GG required by the T7 promoter induced the targeted mutations. This suggests that the RGEN can target any sequence adjacent to an NGG protospacer adjacent motif (PAM) sequence, which occurs once every 8 bp. The off-target alterations at 2 genomic loci harboring double mismatches in the 18-bp targeting sequences were induced in the RGEN-injected embryos. However, we also found that the off-target effects could be reduced by lower dosages of sgRNA. Taken together, our results suggest that CRISPR/Cas-mediated RGENs may be an efficient and flexible tool for genome editing in medaka.