Products Cited in Publications Worldwide Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids

Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids

Nguyen, Long T.; Rananaware, Santosh R.; Yang, Lilia G.; Macaluso, Nicolas C.; Ocana-Ortiz, Julio E.; Meister, Katelyn S.; Pizzano, Brianna L. M.; Sandoval, Luke Samuel W.; Hautamaki, Raymond C.; Fang, Zoe R.; Joseph, Sara M.; Shoemaker, Grace M.; Carman, Dylan R.; Chang, Liwei; Rakestraw, Noah R.; Zachary, Jon F.; Guerra, Sebastian; Perez, Alberto; Jain, Piyush K.

DOI: PMID:

Abstract

CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clin. in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.

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