Ren Xingjie, Yang Zhihao, Xu Jiang, Sun Jin, Mao Decai, Hu Yanhui, Yang Su-Juan, Qiao Huan-Huan, Wang Xia, Hu Qun, Deng Patricia, Liu Lu-Ping, Ji Jun-Yuan, Li Jin Billy, Ni Jian-Quan
Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.
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.
Cell Rep. 2014 Nov 6;9(3):1151-62. doi: 10.1016/j.celrep.2014.09.044. Epub 2014 Oct 23.
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.
CRISPR/Cas9系统最近已成为黑腹果蝇功能基因组学研究的强大工具。然而，影响该系统在果蝇中特异性和效率的单向导RNA（sgRNA）参数仍不清楚。在这里，我们发现与sgRNA有三个或更多核苷酸错配的基因组DNA区域未发生脱靶效应。重要的是，我们记录了诱变效率与六个原间隔相邻基序近端核苷酸（PAMPNs）的sgRNA GC含量之间存在强正相关。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，我们通过同源定向修复使用优化参数产生了HP1a的无效等位基因，并实现了总体诱变率显著高于先前报道。我们的工作展示了sgRNA的全面优化，并有望极大地简化果蝇中的CRISPR/Cas9实验。
