Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.
Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.
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DOI：
10.1016/j.celrep.2014.09.044
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发表时间：
2014-11-06
期刊：
影响因子：
8.8
通讯作者：
Ni JQ
中科院分区：
文献类型：
--
作者：
Ren X;Yang Z;Xu J;Sun J;Mao D;Hu Y;Yang SJ;Qiao HH;Wang X;Hu Q;Deng P;Liu LP;Ji JY;Li JB;Ni JQ
CRISPR/Cas9系统最近成为研究黑腹果蝇功能基因组的有力工具。然而，在果蝇中影响该系统特异性和效率的sgRNA参数仍然不清楚。在这里，我们发现在基因组DNA中与sgRNAs有三个或更多核苷酸错配的区域不会发生脱靶效应。重要的是，我们首次证明了突变效率与六个邻近基序近端核苷酸(PAMPN)的sgRNAGC含量之间的强烈正相关。此外，通过以我们确定的最佳浓度注射精心设计的sgRNA质粒，我们可以一步有效地在四个基因中产生突变。最后，我们用优化的参数通过同源定向修复产生了HP1a的零等位基因，总体突变率明显高于以前报道的突变。我们的工作提供了最全面的sgRNA优化，并有望极大地简化果蝇的CRISPR/Cas9实验。
The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, 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 the first time 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 presents the most comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.
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作者：
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