Housden Benjamin E, Hu Yanhui, Perrimon Norbert
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115;
Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115; Howard Hughes Medical Institute, Harvard Medical School, Boston, Massachusetts 02115.
Cold Spring Harb Protoc. 2016 Sep 1;2016(9):2016/9/pdb.prot090779. doi: 10.1101/pdb.prot090779.
The recent advances in CRISPR-based genome engineering have enabled a plethora of new experiments to study a wide range of biological questions. The major attraction of this system over previous methods is its high efficiency and simplicity of use. For example, whereas previous genome engineering technologies required the generation of new proteins to target each new locus, CRISPR requires only the expression of a different single guide RNA (sgRNA). This sgRNA binds to the Cas9 endonuclease protein and directs the generation of a double-strand break to a highly specific genomic site determined by the sgRNA sequence. In addition, the relative simplicity of the Drosophila genome is a particular advantage, as possible sgRNA off-target sites can easily be avoided. Here, we provide a step-by-step protocol for designing sgRNA target sites using the Drosophila RNAi Screening Center (DRSC) Find CRISPRs tool (version 2). We also describe the generation of sgRNA expression plasmids for the use in cultured Drosophila cells or in vivo. Finally, we discuss specific design requirements for various genome engineering applications.
基于CRISPR的基因组工程技术的最新进展，使得大量新实验得以开展，用于研究广泛的生物学问题。相较于之前的方法，该系统的主要吸引力在于其高效性和易用性。例如，以往的基因组工程技术需要生成新的蛋白质来靶向每个新位点，而CRISPR只需要表达一种不同的单向导RNA（sgRNA）。这种sgRNA与Cas9核酸内切酶蛋白结合，并将双链断裂引导至由sgRNA序列确定的高度特异性基因组位点。此外，果蝇基因组相对简单是一个特别的优势，因为可以很容易地避免sgRNA脱靶位点。在这里，我们提供了一个使用果蝇RNAi筛选中心（DRSC）的Find CRISPRs工具（版本2）设计sgRNA靶位点的分步方案。我们还描述了用于培养的果蝇细胞或体内的sgRNA表达质粒的构建。最后，我们讨论了各种基因组工程应用的具体设计要求。
