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Engineering Highly Thermostable Cas12b via de novo Structural Analyses for One-Pot Detection of Nucleic Acids

45 Pages Posted: 29 Dec 2022 Publication Status: Published

See all articles by Long T. Nguyen

Long T. Nguyen

University of Florida - Department of Chemical Engineering

Santosh R. Rananaware

University of Florida - Department of Chemical Engineering

Lilia G. Yang

University of Florida - Department of Chemical Engineering

Nicolas C. Macaluso

University of Florida - Department of Chemical Engineering

Julio E. Ocana-Ortiz

University of Puerto Rico at Mayagüez - Department of Chemical Engineering

Katelyn S. Meister

University of Florida - Department of Chemical Engineering

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering; University of Florida - Department of Agricultural and Biological Engineering

Luke Samuel W. Sandoval

University of Florida - Department of Biology

Raymond C. Hautamaki

University of Florida - Department of Microbiology and Cell Science

Zoe R. Fang

University of Florida - Department of Microbiology and Cell Science

Sara M. Joseph

University of Florida - Department of Chemical Engineering

Grace M. Shoemaker

University of Florida - Department of Chemical Engineering

Dylan R. Carman

University of Florida - Department of Chemical Engineering

Liwei Chang

University of Florida - Department of Chemistry

Noah R. Rakestraw

University of Florida - Department of Molecular Genetics and Microbiology; University of Florida - Department of Graduate Education

Jon F. Zachary

University of Florida - Department of Graduate Education

Sebastian Guerra

University of Florida - Genetics Institute

Alberto Perez

University of Florida - Department of Chemistry

Piyush K. Jain

University of Florida - Department of Chemical Engineering

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Abstract

CRISPR/Cas-based diagnostics have elevated nucleic acid detection in recent years. The 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 nuclease from Brevibacillus (eBrCas12b) with improved thermostability that falls within the optimal temperature range (60-65oC) of the reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses via DeepDDG and HotSpot Wizard based on Alpha Fold- and SWISS-MODEL-predicted structures, mutations were introduced into the REC and RuvC domains of wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing the thermostability. We expressed, purified, and systematically characterized 49 BrCas12b variants with an emphasis on functionality and thermostability. The assay utilizing eBrCas12b, coined 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 validated SPLENDID clinically in 40 hepatitis C virus (HCV) positive and 40 HCV-negative serum samples, achieving a specificity of 97.5% and an accuracy of 90.0%. Results can be obtained in as little as 20 minutes and with the extraction process, the entire assay can be performed within an hour. We believe that SPLENDID has the potential to become a universal platform for the detection of infectious diseases.

Note:

Funding Information: This work was financially supported by funds from the UF, the UF Herbert Wertheim College of Engineering, Dinesh O. Shah endowed professorship, NIH-NIAID R21AI156321, NIH-NIAID R21AI168795, and NIH-NIGMS R35GM147788.

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, Par Biosciences, and CRIPSR, LLC. The remaining authors declare no competing interests.

Ethics Approval Statement: Patient sample collection and processing were approved by the University of Florida Institutional Review Board (IRB202003085).

Keywords: CRISPR, Cas12b, Protein engineering, Thermal stability, Diagnostics, Hepatitis C, Point-of-care diagnostics, Trans-cleavage, RT-LAMP, One-pot detection

Suggested Citation

Nguyen, Long T. and Rananaware, Santosh R. and Yang, Lilia G. and Macaluso, Nicolas C. and Ocana-Ortiz, Julio E. and Meister, Katelyn S. and Pizzano, Brianna L.M. and Sandoval, Luke Samuel W. and Hautamaki, Raymond C. and Fang, Zoe R. and Joseph, Sara M. and Shoemaker, Grace M. and Carman, Dylan R. and Chang, Liwei and Rakestraw, Noah R. and Zachary, Jon F. and Guerra, Sebastian and Perez, Alberto and Jain, Piyush K., Engineering Highly Thermostable Cas12b via de novo Structural Analyses for One-Pot Detection of Nucleic Acids. Available at SSRN: https://ssrn.com/abstract=4309028 or http://dx.doi.org/10.2139/ssrn.4309028
This version of the paper has not been formally peer reviewed.

Long T. Nguyen

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

Santosh R. Rananaware

University of Florida - Department of Chemical Engineering ( email )

Lilia G. Yang

University of Florida - Department of Chemical Engineering ( email )

Nicolas C. Macaluso

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

Julio E. Ocana-Ortiz

University of Puerto Rico at Mayagüez - Department of Chemical Engineering ( email )

Katelyn S. Meister

University of Florida - Department of Chemical Engineering ( email )

Brianna L.M. Pizzano

University of Florida - Department of Chemical Engineering ( email )

University of Florida - Department of Agricultural and Biological Engineering ( email )

Gainesville, FL
United States

Luke Samuel W. Sandoval

University of Florida - Department of Biology ( email )

Raymond C. Hautamaki

University of Florida - Department of Microbiology and Cell Science ( email )

Zoe R. Fang

University of Florida - Department of Microbiology and Cell Science ( email )

Sara M. Joseph

University of Florida - Department of Chemical Engineering ( email )

Grace M. Shoemaker

University of Florida - Department of Chemical Engineering ( email )

Dylan R. Carman

University of Florida - Department of Chemical Engineering ( email )

Liwei Chang

University of Florida - Department of Chemistry ( email )

Noah R. Rakestraw

University of Florida - Department of Molecular Genetics and Microbiology ( email )

University of Florida - Department of Graduate Education ( email )

Jon F. Zachary

University of Florida - Department of Graduate Education ( email )

Sebastian Guerra

University of Florida - Genetics Institute ( email )

Alberto Perez

University of Florida - Department of Chemistry ( email )

Piyush K. Jain (Contact Author)

University of Florida - Department of Chemical Engineering ( email )

Gainesville, FL
United States

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