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      A CRISPR/Cas12a-Mediated Dual-Mode Electrochemical Biosensor for Polymerase Chain Reaction-Free Detection of Genetically Modified Soybean

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          CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

          CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
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            Nucleic acid detection with CRISPR-Cas13a/C2c2

            Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.
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              The next generation of CRISPR–Cas technologies and applications

              The prokaryote-derived CRISPR–Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research spanning from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologs. Genome editing by CRISPR–Cas can utilize non-homologous end joining (NHEJ) and homologous-directed repair (HDR) for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR–Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, these advances are considerably advancing our understanding of biology and propelling CRISPR–Cas-based tools towards clinical use in gene and cell therapies.

                Author and article information

                Analytical Chemistry
                Anal. Chem.
                American Chemical Society (ACS)
                November 09 2021
                October 26 2021
                November 09 2021
                : 93
                : 44
                : 14885-14891
                [1 ]State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, School of Material Science and Chemical Engineering, Ningbo University, Ningbo 315211, P.R. China
                [2 ]State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R. China
                [3 ]Faculty of Electrical Engineering and Computer Science, Ningbo University, Ningbo 315211, P.R. China
                © 2021






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