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

ABSTRACT: CRISPR-Cas12a (also called Cpf1) has been commonly used for genomic editing, based on its ability to generate precise double-stranded DNA (dsDNA) breaks. Recently, it was demonstrated that Cas12a exhibits unspecific ssDNAse activity upon 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 DNA target sequences corresponding to carbapenemases resistance genes such as KPC, NDM and OXA. Also, with the addition of a reverse-transcription step, we were able to detect viral RNA sequences from DENV, ZIKV and HANTV genomes. 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 were visualized by detection of a fluorescent signal, as well as by the use of a simple lateral flow strip. These results indicate that Cas12a is able to detect both DNA and RNA targets, making it an appropriate and convenient tool to detect all types of pathogens.

SUBMITTER: Curti LA

PROVIDER: S-EPMC7448918 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundCOVID-19 has spread rapidly around the world, affecting a large percentage of the population. When lifting certain mandatory measures for an economic restart, robust surveillance must be established and implemented, with nucleic acid detection for SARS-CoV-2 as an essential component.MethodsWe tried to develop a one-tube detection platform based on RT-RPA (Reverse Transcription and Recombinase Polymerase Isothermal Amplification) and DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR) technology, termed OR-DETECTR, to detect SARS-CoV-2. We designed RT-RPA primers of the RdRp and N genes following the SARS-CoV-2 gene sequence. We optimized reaction components so that the detection process could be carried out in one tube. Specificity was demonstrated by detecting nucleic acid samples from pseudoviruses from seven human coronaviruses and Influenza A (H1N1). Clinical samples were used to validate the platform and all results were compared to rRT-PCR. RNA standards and pseudoviruses diluted by different gradients were used to demonstrate the detection limit. Additionally, we have developed a lateral flow assay based on OR-DETECTR for detecting COVID-19.ResultsThe OR-DETECTR detection process can be completed in one tube, which takes approximately 50 min. This method can specifically detect SARS-CoV-2 from seven human coronaviruses and Influenza A (H1N1), with a low detection limit of 2.5 copies/µl input (RNA standard) and 1 copy/µl input (pseudovirus). Results of six samples from SARS-CoV-2 patients, eight samples from patients with fever but no SARS-CoV-2 infection, and one mixed sample from 40 negative controls showed that OR-DETECTR is 100% consistent with rRT-PCR. The lateral flow assay based on OR-DETECTR can be used for the detection of COVID-19, and the detection limit is 2.5 copies/µl input.ConclusionsThe OR-DETECTR platform for the detection of COVID-19 is rapid, accurate, tube closed, easy-to-operate, and free of large instruments.

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

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