Randomized PAM depletion of AsCas12a. Randomized PAM depletion of AsCas12a

We report the PAMs of AsCas12a using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and AsCas12a-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 AsCas12a. Overall design: Uncleaved DNA from the randomized PAM library was amplified using Illumina Nextera Indices本研究报道了基于无细胞转录翻译（cell-free transcription-translation, TXTL）裂解检测体系的AsCas12a原间隔序列邻近基序（Protospacer Adjacent Motif, PAM）特征。通过向体外反应体系中加入随机PAM文库与AsCas12a向导RNA（gRNA），功能性PAM序列将被裂解，而非功能性PAM序列则得以保留。随后对未被裂解的DNA进行扩增，并借助下一代测序（next-generation sequencing, NGS）分析AsCas12a功能性PAM序列的耗竭情况。整体实验设计：采用Illumina Nextera索引对随机PAM文库中的未裂解DNA进行扩增。