1. Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.
- Author
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Nguyen LT, Rananaware SR, Yang LG, Macaluso NC, Ocana-Ortiz JE, Meister KS, Pizzano BLM, Sandoval LSW, Hautamaki RC, Fang ZR, Joseph SM, Shoemaker GM, Carman DR, Chang L, Rakestraw NR, Zachary JF, Guerra S, Perez A, and Jain PK more...
- Subjects
- Humans, SARS-CoV-2 genetics, COVID-19 Testing, CRISPR-Cas Systems genetics, COVID-19 diagnosis, COVID-19 genetics, Nucleic Acids genetics
- 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 clinically 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., Competing Interests: Declaration of interests L.T.N., S.R.R., and P.K.J. are listed as inventors on the patent applications related to the content of this work. P.K.J. is a co-founder of Genable Biosciences, LLC, Par Biosciences, LLC, and CRISPR, LLC., (Published by Elsevier Inc.) more...
- Published
- 2023
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