Journal
CELL REPORTS
Volume 9, Issue 3, Pages 1151-1162Publisher
CELL PRESS
DOI: 10.1016/j.celrep.2014.09.044
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Funding
- National Basic Research Program (973 Program) [2013CB835100]
- National Natural Science Foundation of China [20131351195, 31301008]
- Specialized Research Fund for the Doctoral Program of Higher Education of China [20121018577, 20120002110056, 20120141120046]
- Natural Science Foundation of Hubei Province [2013CFB031]
- Tsinghua-Peking Center for Life Sciences, an HBUT starting grant [BSQD12142]
- NIH [R01GM102484]
- Ellison Medical Foundation
- Stanford University Department of Genetics
- National Science Foundation
- Stanford Genome Training Program
- Cell and Molecular Biology Training Program
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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.
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