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

doi:10.16288/j.yczz.15-452

遗传 ›› 2016, Vol. 38 ›› Issue (2): 144-154.doi: 10.16288/j.yczz.15-452

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

中国科学院水生生物研究所,淡水生态与生物技术国家重点实验室,武汉 430072

收稿日期:2015-11-04 修回日期:2015-12-28 出版日期:2016-02-20 发布日期:2016-01-26
通讯作者: 孙永华,博士,研究员,研究方向：鱼类发育与生物技术。E-mail: yhsun@ihb.ac.cn
作者简介:张峰华,硕士生,专业方向：鱼类发育与生物技术。E-mail: zfh0606@gmail.com
基金资助:
国家重大科学研究计划项目(编号：2012CB944504),中国科学院重点部署项目(编号：KSZD-EW-Z-001)和国家自然科学基金项目(编号：31222052)资助[Supported by the Major National Scientific Research Projects (No; 2012CB944504), the Key Programs of the Chinese Academy of Sciences (No; KSZD-EW-Z-001) and the National Natural Science Foundation of China (No; 31222052)]

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

Received:2015-11-04 Revised:2015-12-28 Online:2016-02-20 Published:2016-01-26

摘要/Abstract

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

Abstract: Recent years have witnessed the rapid development of the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein(CRISPR/Cas9)system. In order to realize gene knockout with high efficiency 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, including 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.

Key words: CRISPR/Cas9, zebrafish, gene knockout, mutation efficiency

张峰华,王厚鹏,黄思雨,熊凤,朱作言,孙永华. 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[J]. 遗传, 2016, 38(2): 144-154.

Fenghua Zhang, Houpeng Wang, Siyu Huang, Feng Xiong, Zuoyan Zhu, Yonghua Sun. A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos[J]. HEREDITAS(Beijing), 2016, 38(2): 144-154.

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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

A comparison of the knockout efficiencies of two codon-optimized Cas9 coding sequences in zebrafish embryos

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