Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR-Cas12a.
Angew Chem Int Ed Engl
; 63(20): e202403123, 2024 05 13.
Article
en En
| MEDLINE
| ID: mdl-38516796
ABSTRACT
The CRISPR-Cas12a system has emerged as a powerful tool for next-generation nucleic acid-based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA-enabled trans-cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by full-size RNA targets, although LbCas12a exhibits weaker trans-cleavage activity than AsCas12a on both single-stranded DNA and RNA substrates. Remarkably, we find that the RNA-activated Cas12a possesses higher specificity in recognizing mutated target sequences compared to DNA activation. Based on these findings, we develop the "Universal Nuclease for Identification of Virus Empowered by RNA-Sensing" (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM-less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay based on double-stranded DNA activation, demonstrating unrestricted target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a-based nucleic acid detection and further enhance the understanding of CRISPR-Cas biochemistry.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ARN
/
Proteínas Asociadas a CRISPR
/
Sistemas CRISPR-Cas
Límite:
Humans
Idioma:
En
Revista:
Angew Chem Int Ed Engl
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos