Zhang Feng-hua, Wang Hou-peng, Huang Si-yu, Xiong Feng, Zhu Zuo-yan, Sun Yong-hua
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China.
Yi Chuan. 2016 Feb;38(2):144-54. doi: 10.16288/j.yczz.15-452.
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.
近年来，成簇规律间隔短回文重复序列/CRISPR相关蛋白（CRISPR/Cas9）系统发展迅速。为了在斑马鱼中高效且特异地实现基因敲除，多个实验室合成了不同的Cas9 cDNA序列，并将其克隆到不同载体中。在本研究中，我们从两个不同实验室选取了两条常用的经斑马鱼密码子优化的Cas9编码序列（zCas9_bz、zCas9_wc），并利用它们在斑马鱼胚胎中敲除了7个基因，包括外源的egfp和6个内源基因（chd、hbegfa、th、eef1a1b、tyr和tcf7l1a）。我们通过PCR产物直接测序、菌落测序和表型分析，比较了这两条zCas9编码序列的敲除效率。结果表明，在所有条件下，zCas9_wc的敲除效率均高于zCas9_bz。
