The RNA-guided nucleases derived from the CRISPR-Cas systems in bacteria and archaea have found numerous applications in biotechnology, including genome editing, imaging, and gene regulation. However, the discovery of novel Cas nucleases has outpaced their characterization and subsequent exploitation. A key step in characterizing Cas nucleases is determining which protospacer-adjacent motif (PAM) sequences they recognize. Here, we report advances to an in vitro method based on an E. coli cell-free transcription-translation system (TXTL) to rapidly elucidate PAMs recognized by Cas nucleases. The method obviates the need for cloning Cas nucleases or gRNAs, does not require the purification of protein or RNA, and can be performed in less than a day. To advance our previously published method, we incorporated an internal GFP cleavage control to assess the extent of library cleavage as well as Sanger sequencing of the cleaved library to assess PAM depletion prior to next-generation sequencing. We also detail the methods needed to construct all relevant DNA constructs, and how to troubleshoot the assay. We finally demonstrate the technique by determining PAM sequences recognized by the Neisseria meningitidis Cas9, revealing subtle sequence requirements of this highly specific PAM. The overall method offers a rapid means to identify PAMs recognized by diverse CRISPR nucleases, with the potential to greatly accelerate our ability to characterize and harness novel CRISPR nucleases across their many uses.

中文翻译：

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用于破译 CRISPR 原型间隔区相邻基序的详细无细胞转录-翻译分析

源自细菌和古细菌 CRISPR-Cas 系统的 RNA 引导的核酸酶已在生物技术中发现了许多应用，包括基因组编辑、成像和基因调控。然而，新型 Cas 核酸酶的发现已经超过了它们的表征和随后的开发。表征 Cas 核酸酶的一个关键步骤是确定它们识别的原始间隔区相邻基序 (PAM) 序列。在这里，我们报告了基于大肠杆菌无细胞转录-翻译系统 (TXTL) 的体外方法的进展，以快速阐明 Cas 核酸酶识别的 PAM。该方法不需要克隆 Cas 核酸酶或 gRNA，不需要纯化蛋白质或 RNA，并且可以在不到一天的时间内完成。为了推进我们之前发布的方法，我们结合了内部 GFP 切割控制来评估库切割的程度以及切割库的 Sanger 测序，以在下一代测序之前评估 PAM 消耗。我们还详细介绍了构建所有相关 DNA 构建体所需的方法，以及如何对检测进行故障排除。我们最终通过确定脑膜炎奈瑟菌 Cas9 识别的 PAM 序列来展示该技术，揭示了这种高度特异性 PAM 的微妙序列要求。整体方法提供了一种快速方法来识别被不同 CRISPR 核酸酶识别的 PAM，有可能大大加快我们在其多种用途中表征和利用新型 CRISPR 核酸酶的能力。