Programmable RNA detection with CRISPR-Cas12a.
Nat Commun
; 14(1): 5409, 2023 09 05.
Article
em En
| MEDLINE
| ID: mdl-37669948
ABSTRACT
Cas12a, a CRISPR-associated protein complex, has an inherent ability to cleave DNA substrates and is utilized in diagnostic tools to identify DNA molecules. We demonstrate that multiple orthologs of Cas12a activate trans-cleavage in the presence of split activators. Specifically, the PAM-distal region of the crRNA recognizes RNA targets provided that the PAM-proximal seed region has a DNA target. Our method, Split Activator for Highly Accessible RNA Analysis (SAHARA), detects picomolar concentrations of RNA without sample amplification, reverse-transcription, or strand-displacement by simply supplying a short DNA sequence complementary to the seed region. Beyond RNA detection, SAHARA outperforms wild-type CRISPR-Cas12a in specificity towards point-mutations and can detect multiple RNA and DNA targets in pooled crRNA/Cas12a arrays via distinct PAM-proximal seed DNAs. In conclusion, SAHARA is a simple, yet powerful nucleic acid detection platform based on Cas12a that can be applied in a multiplexed fashion and potentially be expanded to other CRISPR-Cas enzymes.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
RNA
/
Sistemas CRISPR-Cas
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2023
Tipo de documento:
Article