title: A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.
abstract: CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs.
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Haiwei
authors.initials: H
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Xixi
authors.initials: X
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Cunxian
authors.initials: C
authors.lastname: Fan
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China; Department of Anesthesiology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Xie
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Chengxian
authors.initials: C
authors.lastname: Xu
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Yongbo
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
authors.forename: Xiaoxia
authors.initials: X
authors.lastname: Wu
authors.affiliation: Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Graduate School of the Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, China. Electronic address: hbzhang@sibs.ac.cn.
authors.forename: Haibing
authors.initials: H
authors.lastname: Zhang
mesh_terms: D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases; D055786:Gene Knockout Techniques; D005818:Genetic Engineering; D057809:HEK293 Cells; D019073:HT29 Cells; D006801:Humans; D016086:Lentivirus; D008156:Luciferases / Q000737:chemistry; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics / Q000302:isolation & purification*; D012641:Selection, Genetic; D014162:Transfection
chemical_list: D001426:Bacterial Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D008156:Luciferases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: CRISPR/Cas9; Cell death; Gene knockout; Necroptosis; sgRNA selection