CRISPR-based platform for carbapenemases and emerging viruses detection using Cas12a (Cpf1) effector nuclease

CURTI, LUCIA ANA; PEREYRA-BONNET, FEDERICO; REPIZO, GUILLERMO DANIEL; FAY, JESSICA VANNINA; SALVATIERRA, KARINA; BLARIZA, MARÍA JOSÉ; IBAÑEZ-ALEGRE, DAIANA; RINFLERCH, ADRIANA RAQUEL; MIRETTI, MARCOS; GIMENEZ, CARLA ALEJANDRA

Emerging Microbes & Infections

Taylor AND Francis

Lugar: Londres; Año: 2020 vol. 9 p. 1140 - 1148

CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precisedouble-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activityupon target recognition. This feature allows CRISPR-Cas to be coupled with a ssDNA reporter and generate a fast,accurate and ultrasensitive molecular detection method. Here, we demonstrate that Cas12a was able to detect DNAtarget sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with theaddition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTVgenomes. In all cases, assay run time was less than two hours. Additionally, we report attomolar levels of detection.This methodology was validated using clinical samples from patients infected with Dengue virus. Reactions werevisualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicatethat Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect alltypes of pathogens.