RESUMEN
Early detection of viral pathogens by DNA-sensors in clinical samples, contaminated foods, soil or water can dramatically improve clinical outcomes and reduce the socioeconomic impact of diseases such as COVID-19. Clustered regularly interspaced short palindromic repeat (CRISPR) and its associated protein Cas12a (previously known as CRISPR-Cpf1) technology is an innovative new-generation genomic engineering tool, also known as 'genetic scissors', that has demonstrated the accuracy and has recently been effectively applied as appropriate (E-CRISPR) DNA-sensor to detect the nucleic acid of interest. The CRISPR-Cas12a from Prevotella and Francisella 1 are guided by a short CRISPR RNA (gRNA). The unique simultaneous cis- and trans- DNA cleavage after target sequence recognition at the PAM site, sticky-end (5-7 bp) employment, and ssDNA/dsDNA hybrid cleavage strategies to manipulate the attractive nature of CRISPR-Cas12a are reviewed. DNA-sensors based on the CRISPR-Cas12a technology for rapid, robust, sensitive, inexpensive, and selective detection of virus DNA without additional sample purification, amplification, fluorescent-agent- and/or quencher-labeling are relevant and becoming increasingly important in industrial and medical applications. In addition, CRISPR-Cas12a system shows great potential in the field of E-CRISPR-based bioassay research technologies. Therefore, we are highlighting insights in this research direction.
Asunto(s)
Sistemas CRISPR-Cas/fisiología , ADN Viral/aislamiento & purificación , Técnicas de Amplificación de Ácido Nucleico , Animales , Técnicas Biosensibles/métodos , Técnicas Biosensibles/tendencias , COVID-19/virología , ADN Viral/análisis , Contaminantes Ambientales/análisis , Contaminantes Ambientales/aislamiento & purificación , Contaminación de Alimentos/análisis , Humanos , Tipificación Molecular/métodos , Tipificación Molecular/tendencias , Técnicas de Amplificación de Ácido Nucleico/métodos , Técnicas de Amplificación de Ácido Nucleico/tendencias , SARS-CoV-2/genética , Virología/métodos , Virología/tendencias , Virosis/clasificación , Virosis/diagnóstico , Virosis/virologíaRESUMEN
The limitations of conventional diagnostic procedures, such as real-time PCR-based methods and serological tests, have led the scientific community to innovate alternative nucleic acid detection approaches for SARS-CoV-2 RNA, thereby addressing the dire need for increased testing. Such approaches aim to provide rapid, accurate, cost-effective, sensitive, and high-throughput detection of SARS-CoV-2 RNA, on multiple specimen types, and without specialized equipment and expertise. The CRISPR-Cas13 system functions as a sequence-specific RNA-sensing tool that has recently been harnessed to develop simplified and flexible testing formats. This review recapitulates technical advances in the most recent CRISPR-Cas13-based methods for SARS-CoV-2/COVID-19 diagnosis. The challenges and opportunities for implementing mass testing using these novel CRISPR-Cas13 platforms are critically analyzed.