两种Cas9编码基因在斑马鱼胚胎中基因敲除效率的比较.pdf

Hereditas (Beijing) 2016 年*月, 38(*): 1―11 研究报告 两种密码子优化的Cas9 编码基因在斑马鱼胚胎中基因 敲除效率的比较 张峰华，王厚鹏，黄思雨，熊凤，朱作言，孙永华 430072 摘要: 为在斑马鱼中获得特异且高效的基因敲除，多个实验室独立人工合成了序列彼此不一的Cas9 cDNA 序 列，并克隆入不同的体外转录载体。本文选取两种斑马鱼密码子优化的Cas9 编码序列(zCas9_bz 和zCas9_wc) ， 对斑马鱼胚胎中的7 个基因(外源egfp 及内源chd 、hbegfa 、th 、eef1a1b 、tyr 、tcf7l1a)分别进行敲除，通过PCR 产物测序、克隆测序和表型分析比较了两种Cas9 的敲除效率。结果发现，zCas9_wc 在各种情况下都显现出较 高的敲除效率，而zCas9_bz 的效率相对较低。 关键词: CRISPR/Cas9 ；斑马鱼；基因敲除；突变效率 A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos Fenghua Zhang, Houpeng Wang, Siyu Huang, Feng Xiong, Zuoyan Zhu, Yonghua Sun State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China Abstract: Recent years have witnessed the rapid development of the clustered regularly interspaced short palin- dromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high effi- ciency and specificity in zebrafish, several labs have synthesized distinct Cas9 cDNA sequences which were cloned into different vectors. In this study, we chose two commonly used zebrafish-codon-optimized Cas9 coding sequences (zCas9_bz , zCas9_wc) from two different labs, and utilized them to knockout seven genes in zebrafish embryos, in- cluding the exogenous egfp and six endogenous genes (chd, hbegfa, th, eef1a1b, tyr and tcf7l1a). We compared the knockout efficiencies resulting from the two zCas9 coding sequences, by direct sequencing of PCR products, colony sequencing and phenotypic analysis. The results showed that the knockout efficiency of zCas9_wc was higher than that of zCas9_bz in all conditions. Keywords: CRISPR/Cas9; zebrafish; gene knockout; mutation efficiency 收稿日期: 20151104; 修回日期: 20161228 基金项目: (2012CB944504)(KSZD-EW-Z-001)(