Sun Yonghua, Zhang Bo, Luo Lingfei, Shi De-Li, Wang Han, Cui Zongbin, Huang Honghui, Cao Ying, Shu Xiaodong, Zhang Wenqing, Zhou Jianfeng, Li Yun, Du Jiulin, Zhao Qingshun, Chen Jun, Zhong Hanbing, Zhong Tao P, Li Li, Xiong Jing-Wei, Peng Jinrong, Xiao Wuhan, Zhang Jian, Yao Jihua, Yin Zhan, Mo Xianming, Peng Gang, Zhu Jun, Chen Yan, Zhou Yong, Liu Dong, Pan Weijun, Zhang Yiyue, Ruan Hua, Liu Feng, Zhu Zuoyan, Meng Anming
State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, Hubei, 430072, China.
Key Laboratory of Cell Proliferation and Differentiation of the Ministry of Education, Peking University Genome Editing Research Center, College of Life Sciences, Peking University, Beijing, 100871, China.
Genome Res. 2019 Dec 12;30(1):118-26. doi: 10.1101/gr.248559.119.
Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.
成熟的CRISPR/Cas9技术介导的基因组编辑极大地促进了我们对许多生物学过程的理解。然而，对任何物种或模式生物进行完整的全基因组敲除却鲜有成功。在此，我们对斑马鱼1号染色体上的所有基因（1333个）进行了系统性敲除，成功突变了1029个基因，并产生了对应于636个基因的1039个可种系传递的等位基因。同时，通过高通量生物信息学分析，我们发现序列特征在有效gRNA靶向特定目的基因中起关键作用，而基因靶向成功率与靶位点的GC含量呈正相关。此外，我们发现所有突变体中近四分之一与人类疾病相关，并在此描述了几个具有代表性的CRISPR/Cas9产生的突变体。此外，我们试图确定导致基因突变体与反义吗啉代介导的敲低胚胎之间出现不同表型的潜在机制。总之，这项工作通过CRISPR/Cas9技术产生了首个全染色体范围的斑马鱼基因突变体集合，这将成为该领域的宝贵资源，我们的生物信息学分析也为设计用于成功基因编辑的基因特异性gRNA提供了一些有用的指导。
