title: Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.
abstract: The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
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; Tsinghua Fly Center, 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.
authors.forename: Jiang
authors.initials: J
authors.lastname: Xu
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Jin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China; Sichuan Academy of Grassland Science, Chengdu 611731, China.
authors.forename: Decai
authors.initials: D
authors.lastname: Mao
authors.affiliation: Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
authors.forename: Yanhui
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
authors.forename: Su-Juan
authors.initials: SJ
authors.lastname: Yang
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: Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
authors.forename: Qun
authors.initials: Q
authors.lastname: Hu
authors.affiliation: Department of Genetics, Stanford University, Stanford, CA 94305, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Deng
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, TX 77843, USA.
authors.forename: Jun-Yuan
authors.initials: JY
authors.lastname: Ji
authors.affiliation: Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address: jin.billy.li@stanford.edu.
authors.forename: Jin Billy
authors.initials: JB
authors.lastname: Li
authors.affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address: nijq@mail.tsinghua.edu.cn.
authors.forename: Jian-Quan
authors.initials: JQ
authors.lastname: Ni
mesh_terms: D000483:Alleles; D000818:Animals; D001482:Base Composition / Q000235:genetics; D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D002868:Chromosomal Proteins, Non-Histone / Q000235:genetics; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004260:DNA Repair / Q000235:genetics; D004331:Drosophila melanogaster / Q000235:genetics*; D005854:Germ Cells / Q000378:metabolism; D040582:Inheritance Patterns / Q000235:genetics; D007267:Injections; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000235:genetics; D009154:Mutation / Q000235:genetics; D059645:Mutation Rate; D009711:Nucleotides / Q000235:genetics; D009928:Organ Specificity; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000378:metabolism*
chemical_list: D064130:CRISPR-Associated Proteins; D002868:Chromosomal Proteins, Non-Histone; D009711:Nucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D000090266:Chromobox Protein Homolog 5