title: Performance of the Cas9 nickase system in Drosophila melanogaster.
abstract: Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Xingjie
authors.initials: X
authors.lastname: Ren
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Zhihao
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Decai
authors.initials: D
authors.lastname: Mao
authors.affiliation: School of Life Sciences, Tsinghua University, Beijing 100084, China.
authors.forename: Zai
authors.initials: Z
authors.lastname: Chang
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Huan-Huan
authors.initials: HH
authors.lastname: Qiao
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Xia
authors.initials: X
authors.lastname: Wang
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Cui
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
authors.forename: Lu-Ping
authors.initials: LP
authors.lastname: Liu
authors.affiliation: Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, Texas 77843.
authors.forename: Jun-Yuan
authors.initials: JY
authors.lastname: Ji
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China School of Basic Medical Sciences, Wuhan University, Wuhan 430071, China College of Bioengineering, Hubei University of Technology, Wuhan 430068, China jiangxu@mail.tsinghua.edu.cn nijq@mail.tsinghua.edu.cn.
authors.forename: Jiang
authors.identifier: http://orcid.org/0000-0002-6933-5453
authors.initials: J
authors.lastname: Xu
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China jiangxu@mail.tsinghua.edu.cn nijq@mail.tsinghua.edu.cn.
authors.forename: Jian-Quan
authors.initials: JQ
authors.lastname: Ni
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified / Q000378:metabolism; D060565:Argonaute Proteins / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics; D004260:DNA Repair; D003850:Deoxyribonuclease I / Q000235:genetics / Q000378:metabolism*; D029721:Drosophila Proteins / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology* / Q000254:growth & development; D004625:Embryo, Nonmammalian / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D016296:Mutagenesis; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism
chemical_list: D060565:Argonaute Proteins; D029721:Drosophila Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; C116498:piwi protein, Drosophila; D003850:Deoxyribonuclease I
keywords: CRISPR; Cas9; nickase; off-target; piwi