![]() (28,29) Recently, substantial efforts using computational approaches for metagenomic mining resulted in the discovery of novel CRISPR/Cas systems, including class II/type VI Cas proteins that exclusively target ssRNA substrates. (24)ĬRISPR/Cas systems possess great potential for various applications, so researchers continuously search for, identify, and characterize new Cas effectors to increase utility and develop new tools for in vivo and in vitro applications. (21−23) To bypass the need for nucleic acid purification, HUDSON (Heating Unextracted Diagnostic Samples to Obliterate Nucleases) was developed and coupled with SHERLOCK. For example, the CRISPR-Cas13 system coupled with RPA is used for virus detection via SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing). (19,20) This collateral cleavage activity of Cas12 and Cas13 has been harnessed for nucleic acid detection. Once a complementary sequence is recognized, the enzyme cleaves the nucleic acid in cis and, once activated by the initial recognition, exhibits collateral trans cleavage activities, cleaving single-stranded (ss) DNA or RNA molecules present in the reaction. (12−18) The Cas12 and Cas13 enzymes complex with their corresponding single-guide RNA (sgRNA) and scan a DNA or RNA template for a complementary sequence. (3,11) Recently, RT-LAMP and RT-RPA were coupled with CRISPR Cas9, Cas12, and Cas13 enzymes for the sensitive and specific detection of nucleic acids and viruses, including SARS-CoV-2. (9,10) LAMP and RPA are cost-effective, field-deployable, and scalable alternatives to PCR-based methods. To overcome requirements for complex equipment, researchers have developed technologies for a single-step, isothermal amplification of nucleic acids, including loop-mediated isothermal amplification (LAMP) and recombinase polymerase amplification (RPA). Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. ![]() Our system exhibits sensitivity and specificity comparable to other CRISPR systems. ![]() We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity identifying new enzymes may improve CRISPR-based diagnostics. These detection platforms take advantage of CRISPR enzymes’ RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. ![]()
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