Randomized PAM depeletion of NmeCas9. Randomized PAM depeletion of NmeCas9

We report the PAMs of NmeCas9 using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and NmeCas9-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of NmeCas9. Overall design: Pam determination assay for NmeCas9本研究基于无细胞转录翻译偶联（Transcription-translation coupled, TXTL）裂解检测实验，报道了NmeCas9的原间隔序列邻近基序（Protospacer Adjacent Motif, PAM）。通过体外添加随机化PAM文库与NmeCas9-向导RNA（guide RNA, gRNA）复合物，具备功能的PAM序列会被切割，而非功能PAM则得以保留。通过扩增未被切割的DNA，我们利用下一代测序（Next-Generation Sequencing, NGS）分析NmeCas9功能PAM的耗竭情况。实验整体设计：NmeCas9的PAM鉴定检测实验