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Abstract：
Many CRISPR-Cas immune systems generate guide (g)RNAs using trans-activating CRISPR RNAs (tracrRNAs). Recent work revealed that Cas9 tracrRNAs could be reprogrammed to convert any RNA-of-interest into a gRNA, linking the RNA\'s presence to Cas9-mediated cleavage of double-stranded (ds)DNA. Here, we reprogram tracrRNAs from diverse Cas12 nucleases, linking the presence of an RNA-of-interest to dsDNA cleavage and subsequent collateral single-stranded DNA cleavage-all without the RNA necessarily encoding a protospacer-adjacent motif (PAM). After elucidating nuclease-specific design rules, we demonstrate PAM-independent RNA detection with Cas12b, Cas12e, and Cas12f nucleases. Furthermore, rationally truncating the dsDNA target boosts collateral cleavage activity, while the absence of a gRNA reduces background collateral activity and enhances sensitivity. Finally, we apply this platform to detect 16 S rRNA sequences from five different bacterial pathogens using a universal reprogrammed tracrRNA. These findings extend tracrRNA reprogramming to diverse dsDNA-targeting Cas12 nucleases, expanding the flexibility and versatility of CRISPR-based RNA detection.
摘要：
许多CRISPR-Cas免疫系统使用反式激活CRISPRRNA(tracrRNA)产生指导(g)RNA。最近的研究表明，Cas9tracrRNA可以重新编程，将任何感兴趣的RNA转化为gRNA，将RNA的存在与Cas9介导的双链(DS)DNA切割连接。这里,我们重新编程来自不同Cas12核酸酶的tracrRNA，将目的RNA的存在与dsDNA切割和随后的附带单链DNA切割连接-所有这些都不需要RNA编码前间隔区相邻基序（PAM）。在阐明了核酸酶特定的设计规则之后，我们证明了用Cas12b检测PAM非依赖性RNA，Cas12e,和Cas12f核酸酶。此外，合理地截断dsDNA靶增强了附带切割活性，而gRNA的缺失会降低背景侧支活性并增强敏感性。最后,我们使用通用重编程的tracrRNA应用该平台检测来自五种不同细菌病原体的16SrRNA序列。这些发现将tracrRNA重编程扩展到不同的dsDNA靶向Cas12核酸酶，扩大基于CRISPR的RNA检测的灵活性和多功能性。
