CHE: Engineered CRISPR/Cas systems as next generation diagnostics & gene editing tools

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Title: Engineered CRISPR/Cas systems as next generation diagnostics & gene editing tools

Abstract: Type V and VI CRISPR/Cas systems have emerged as promising cutting-edge diagnostic tools for nucleic acid detection. Nevertheless, the challenge of low target concentrations necessitates pre-amplification, which constrains their practical use in point-of-care (POC) settings. Our lab has explored multiple strategies to create a platform for detecting nucleic acids that is as quick and simple as rapid antigen tests but has an accuracy of a qPCR test.
Firstly, in pursuit of a unified testing method that combines RT-LAMP and CRISPR at a single operating temperature, we delved into various thermophilic Cas12b orthologs. This exploration led to the discovery of BrCas12b, which possessed exceptional thermostability and enabled the development of a one-pot assay known as SPADE. SPADE successfully identified 5 major SARS-CoV-2 variants at temperatures between 60-62°C. Secondly, employing a combination of structural design and machine learning techniques, we engineered BrCas12b to further enhance its thermophilicity, resulting in the creation of a robust one-pot assay named SPLENDID. The SPLENDID effectively operates within the desirable temperature range of 65-67°C and demonstrates remarkable accuracy in detecting HCV genotype 1 and SARS-CoV-2 RNA clinically.
Thirdly, our research unveiled various CRISPR/Cas12a orthologs capable of partially recognizing native RNAs. This discovery paved the way for the development of SAHARA, a split activator system designed for target RNA detection using CRISPR/Cas12a without the need for reverse transcription. SAHARA represents an amplification-free and reverse-transcription-free one-pot detection technology for the direct analysis of RNAs. Furthermore, our laboratory has been actively investigating methods to develop PAM-less approaches for detecting nucleic acids and single-point mismatches without constraints. These groundbreaking advancements hold the potential to revolutionize the next generation of diagnostic tools, effectively addressing the pressing need for rapid, accurate, and accessible point-of-care tests for infectious diseases.

Bio: Dr. Jain received a bachelor’s in pharmacy from India, followed by an interdisciplinary Ph.D. at the University of Missouri-Kansas City, where he pioneered light-responsive biochemical tools. Subsequently, as a postdoc at MIT in Prof. Sangeeta Bhatia’s lab, Dr. Jain continued to develop light-responsive nanosensors and gene-editing technologies. As a Shah Rising Star Professor at UF, Dr. Jain’s team is developing next-generation diagnostics and genome-editing technologies. His work is routinely published in high-impact journals and has been highlighted by over 10,000 news websites including a TEDx talk. He has over 30 pending or issued patents and co-founded multiple startups. His research endeavors have been generously funded, with his projects securing 12 grants including from NIH, CDC, and USDA, raising substantial funding of up to ~$15 million. Dr. Jain has been honored with the Shah Rising Star Professorship, the UF Provost Excellence Award for Assistant Professors, and the MIRA-ESI award (R35) by the NIH-NIGMS.