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Cell Rep. 2014 Nov 6;9(3):1151-62. doi: 10.1016/j.celrep.2014.09.044. Epub 2014 Oct 23.

Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.

Author information

1
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
2
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.
3
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China; Sichuan Academy of Grassland Science, Chengdu 611731, China.
4
Department of Genetics, Harvard Medical School, Boston, MA 02115, USA.
5
Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
6
Department of Genetics, Stanford University, Stanford, CA 94305, USA.
7
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China; Tsinghua Fly Center, Tsinghua University, Beijing 100084, China.
8
Department of Molecular and Cellular Medicine, College of Medicine, Texas A&M Health Science Center, College Station, TX 77843, USA.
9
Department of Genetics, Stanford University, Stanford, CA 94305, USA. Electronic address: jin.billy.li@stanford.edu.
10
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China. Electronic address: nijq@mail.tsinghua.edu.cn.

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.

PMID:
25437567
PMCID:
PMC4250831
DOI:
10.1016/j.celrep.2014.09.044
[Indexed for MEDLINE]
Free PMC Article

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