Walton Russell T, Hsu Jonathan Y, Joung J Keith, Kleinstiver Benjamin P
Center for Genomic Medicine, Massachusetts General Hospital, Boston, MA, USA.
Department of Pathology, Massachusetts General Hospital, Boston, MA, USA.
Nat Protoc. 2021 Mar;16(3):1511-1547. doi: 10.1038/s41596-020-00465-2. Epub 2021 Feb 5.
The continued expansion of the genome-editing toolbox necessitates methods to characterize important properties of CRISPR-Cas enzymes. One such property is the requirement for Cas proteins to recognize a protospacer-adjacent motif (PAM) in DNA target sites. The high-throughput PAM determination assay (HT-PAMDA) is a method that enables scalable characterization of the PAM preferences of different Cas proteins. Here, we provide a step-by-step protocol for the method, discuss experimental design considerations, and highlight how the method can be used to profile naturally occurring CRISPR-Cas9 enzymes, engineered derivatives with improved properties, orthologs of different classes (e.g., Cas12a), and even different platforms (e.g., base editors). A distinguishing feature of HT-PAMDA is that the enzymes are expressed in a cell type or organism of interest (e.g., mammalian cells), permitting scalable characterization and comparison of hundreds of enzymes in a relevant setting. HT-PAMDA does not require specialized equipment or expertise and is cost effective for multiplexed characterization of many enzymes. The protocol enables comprehensive PAM characterization of dozens or hundreds of Cas enzymes in parallel in <2 weeks.
基因组编辑工具箱的持续扩展需要有方法来表征CRISPR-Cas酶的重要特性。其中一个特性是Cas蛋白识别DNA靶位点中前间隔序列邻近基序（PAM）的要求。高通量PAM测定法（HT-PAMDA）是一种能够对不同Cas蛋白的PAM偏好进行可扩展表征的方法。在此，我们提供该方法的详细步骤方案，讨论实验设计考量因素，并强调该方法如何用于分析天然存在的CRISPR-Cas9酶、具有改进特性的工程衍生物、不同类型的直系同源物（如Cas12a），甚至不同平台（如碱基编辑器）。HT-PAMDA的一个显著特点是酶在感兴趣的细胞类型或生物体（如哺乳动物细胞）中表达，从而能够在相关环境中对数百种酶进行可扩展的表征和比较。HT-PAMDA不需要专门的设备或专业知识，对于多种酶的多重表征而言成本效益高。该方案能够在不到两周的时间内并行对数十种或数百种Cas酶进行全面的PAM表征。
