Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, OK, United States.
Department of Chemistry and Biochemistry, Price Family Foundation Institute of Structural Biology, Stephenson Life Sciences Research Center, University of Oklahoma, Norman, OK, United States.
Methods Enzymol. 2023;679:97-129. doi: 10.1016/bs.mie.2022.08.048.
The CRISPR-associated (Cas) Cas12a is the effector protein for type V-A CRISPR systems. Cas12a is a sequence-specific endonuclease that targets and cleaves DNA containing a cognate short signature motif, called the protospacer adjacent motif (PAM), flanked by a 20 nucleotide (nt) segment that is complementary to the "guide" region of its CRISPR RNA (crRNA). The guide sequence of the crRNA can be programmed to target any DNA with a cognate PAM and is the basis for Cas12a's current use for gene editing in numerous organisms and for medical diagnostics. While Cas9 (type II effector protein) is widely used for gene editing, Cas12a possesses favorable features such as its smaller size and creation of staggered double-stranded DNA ends after cleavage that enhances cellular recombination events. Collected here are protocols for the recombinant purification of Cas12a and the transcription of its corresponding programmable crRNA that are used in a variety of Cas12a-specific in vitro activity assays such as the cis, the trans and the guide-RNA independent DNA cleavage activities with multiple substrates. Correspondingly, protocols are included for the quantification of the activity assay data using ImageJ and the use of MATLAB for rate constant calculations. These procedures can be used for further structural and mechanistic studies of Cas12a orthologs and other Cas proteins.
CRISPR 相关（Cas）Cas12a 是 V-A 型 CRISPR 系统的效应蛋白。Cas12a 是一种序列特异性内切酶，靶向并切割含有同源短签名基序（称为前导邻近基序（PAM）的 DNA），侧翼为 20 个核苷酸（nt）片段，与其 CRISPR RNA（crRNA）的“向导”区域互补。crRNA 的向导序列可被编程靶向任何具有同源 PAM 的 DNA，这是 Cas12a 当前在许多生物体中的基因编辑和医学诊断中应用的基础。虽然 Cas9（II 型效应蛋白）被广泛用于基因编辑，但 Cas12a 具有一些有利的特征，例如其体积较小，并且在切割后产生交错的双链 DNA 末端，从而增强细胞重组事件。这里收集了 Cas12a 的重组纯化以及其相应可编程 crRNA 转录的方案，这些方案用于各种 Cas12a 特异性体外活性测定，例如 cis、trans 和向导-RNA 独立的 DNA 切割活性，使用多种底物。相应地，还包括使用 ImageJ 量化活性测定数据的方案，以及使用 MATLAB 进行速率常数计算的方案。这些程序可用于 Cas12a 同源物和其他 Cas 蛋白的进一步结构和机制研究。
