RESUMEN

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.

Asunto(s)

Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , Proteínas Asociadas a CRISPR/metabolismo , Proteínas Asociadas a CRISPR/genética , ARN Ribosómico 16S/genética , ARN Ribosómico 16S/metabolismo , ADN/metabolismo , ADN/genética , ARN/metabolismo , ARN/genética , División del ADN , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Edición Génica/métodos , Endodesoxirribonucleasas/metabolismo , Endodesoxirribonucleasas/genética , Francisella/genética