Anders Carolin, Niewoehner Ole, Jinek Martin
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Methods Enzymol. 2015;558:515-537. doi: 10.1016/bs.mie.2015.02.008. Epub 2015 Mar 12.
The programmable RNA-guided DNA cleavage activity of the bacterial CRISPR-associated endonuclease Cas9 is the basis of genome editing applications in numerous model organisms and cell types. In a binary complex with a dual crRNA:tracrRNA guide or single-molecule guide RNA, Cas9 targets double-stranded DNAs harboring sequences complementary to a 20-nucleotide segment in the guide RNA. Recent structural studies of the enzyme have uncovered the molecular mechanism of RNA-guided DNA recognition. Here, we provide protocols for electrophoretic mobility shift and fluorescence-detection size exclusion chromatography assays used to probe DNA binding by Cas9 that allowed us to reconstitute and crystallize the enzyme in a ternary complex with a guide RNA and a bona fide target DNA. The procedures can be used for further mechanistic investigations of the Cas9 endonuclease family and are potentially applicable to other multicomponent protein-nucleic acid complexes.
细菌CRISPR相关的核酸内切酶Cas9的可编程RNA引导的DNA切割活性是众多模式生物和细胞类型中基因组编辑应用的基础。在与双crRNA:tracrRNA向导或单分子向导RNA形成的二元复合物中，Cas9靶向双链DNA，这些双链DNA含有与向导RNA中20个核苷酸片段互补的序列。最近对该酶的结构研究揭示了RNA引导的DNA识别的分子机制。在这里，我们提供了用于电泳迁移率变动分析和荧光检测尺寸排阻色谱分析的方案，这些分析用于探测Cas9与DNA的结合，使我们能够在与向导RNA和真正的靶DNA形成的三元复合物中重构并结晶该酶。这些程序可用于对Cas9核酸内切酶家族进行进一步的机制研究，并可能适用于其他多组分蛋白质-核酸复合物。
