Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9
- Yonghua Sun1,
- Bo Zhang2,
- Lingfei Luo3,
- De-Li Shi4,
- Han Wang5,
- Zongbin Cui1,
- Honghui Huang3,
- Ying Cao6,
- Xiaodong Shu7,
- Wenqing Zhang8,
- Jianfeng Zhou9,
- Yun Li9,
- Jiulin Du10,
- Qingshun Zhao11,
- Jun Chen12,
- Hanbing Zhong13,
- Tao P. Zhong14,
- Li Li3,
- Jing-Wei Xiong15,
- Jinrong Peng12,
- Wuhan Xiao1,
- Jian Zhang16,
- Jihua Yao17,
- Zhan Yin1,
- Xianming Mo18,
- Gang Peng19,
- Jun Zhu20,
- Yan Chen21,
- Yong Zhou22,
- Dong Liu13,
- Weijun Pan22,
- Yiyue Zhang8,
- Hua Ruan3,
- Feng Liu23,
- Zuoyan Zhu1,
- Anming Meng24 and
- The ZAKOC Consortium25
- 1State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Innovation Academy for Seed Design, Chinese Academy of Sciences, Wuhan, Hubei, 430072, China;
- 2Key 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;
- 3School of Life Sciences, Southwest University, Chongqing, 400715, China;
- 4Guangdong Medical University, Zhanjiang, Guangdong, 524023, China;
- 5Center for Circadian Clocks, Soochow University, Suzhou, Jiangsu, 215123, China;
- 6School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China;
- 7Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, 510530, China;
- 8Division of Cell, Developmental and Integrative Biology, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 510006, China;
- 9School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong, 266100, China;
- 10State Key Laboratory of Neuroscience, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China;
- 11Model Animal Research Center, Nanjing University, Nanjing, Jiangsu, 210093, China;
- 12College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China;
- 13Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, 518055, China;
- 14Institute of Biomedical Sciences, East China Normal University, Shanghai, 200062, China;
- 15College of Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, 100871, China;
- 16School of Life Sciences, Yunnan University, Kunming, Yunnan, 650091, China;
- 17School of Life Sciences, Fudan University, Shanghai, 200433, China;
- 18State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China;
- 19Institutes of Brain Science, Fudan University, Shanghai, 200433, China;
- 20Sino-French Research Center for Life Sciences and Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China;
- 21Institute of Health Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China;
- 22CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai, 200031, China;
- 23State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China;
- 24School of Life Sciences, Tsinghua University, Beijing, 100084, China;
- 26State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, Innovation Academy of Seed Design, Chinese Academy of Sciences, Wuhan, Hubei, 430072, China;
- 27Key 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;
- 28School of Life Sciences, Southwest University, Chongqing, 400715, China;
- 29School of Life Sciences, Shandong University, Qingdao, 266237, China;
- 30Center for Circadian Clocks, Soochow University, Suzhou, Jiangsu, 215123, China;
- 31School of Life Sciences and Technology, Tongji University, Shanghai, 200092, China;
- 32Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, Guangdong, 510530, China;
- 33Division of Cell, Developmental and Integrative Biology, School of Medicine, South China University of Technology, Guangzhou, Guangdong, 510006, China;
- 34School of Medicine and Pharmacy, Ocean University of China, Qingdao, Shandong, 266100, China;
- 35State Key Laboratory of Neuroscience, Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, 200031, China;
- 36Model Animal Research Center, Nanjing University, Nanjing, Jiangsu, 210093, China;
- 37College of Life Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China;
- 38Department of Biology, South University of Science and Technology of China, Shenzhen, Guangdong, 518055, China;
- 39Institute of Chinese Medical Sciences, University of Macau, Macau, China;
- 40Institute of Biomedical Sciences, Shanghai Key Laboratory of Regulatory Biology, East China Normal University, Shanghai, 200062, China;
- 41College of Life Sciences, Institute of Molecular Medicine, Peking University, Beijing, 100871, China;
- 42College of Animal Sciences, Zhejiang University, Hangzhou, Zhejiang, 310058, China;
- 43State Key Laboratory for Conservation and Utilization of Bio-Resources, Kunming, 650091, China;
- 44Center for Life Sciences, School of Life Sciences, Yunnan University, Kunming, 650091, China;
- 45State Key Laboratory of Molecular Developmental Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, 100101, China;
- 46School of Life Sciences, Fudan University, Shanghai, 200433, China;
- 47State Key Laboratory of Biotherapy, West China Hospital, Sichuan University, Chengdu, Sichuan, 610041, China;
- 48Institutes of Brain Science, Fudan University, Shanghai, 200433, China;
- 49Sino-French Research Center for Life Sciences and Genomics, Rui-Jin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China;
- 50Institute of Health Sciences, Chinese Academy of Sciences & Shanghai Jiao Tong University School of Medicine, Shanghai, 200025, China;
- 51CAS Key Laboratory of Nutrition, Metabolism and Food Safety, Shanghai Institute of Nutrition and Health, Chinese Academy of Sciences, Shanghai, 200031, China;
- 52State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, Beijing, 100101, China;
- 53School of Life Sciences, Tsinghua University, Beijing, 100084, China;
- 54Present address: Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China
Abstract
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.
Footnotes
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↵25 A complete list of the ZAKOC Consortium authors appears at the end of this paper.
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.248559.119.
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Freely available online through the Genome Research Open Access option.
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54 Present address: Affiliated Hospital of Guangdong Medical University, Zhanjiang, 524001, China
- Received January 22, 2019.
- Accepted November 6, 2019.
This article, published in Genome Research, is available under a Creative Commons License (Attribution 4.0 International), as described at http://creativecommons.org/licenses/by/4.0/.
