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Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

View ORCID ProfileLong T. Nguyen, View ORCID ProfileBrianna M. Smith, View ORCID ProfilePiyush K. Jain
doi: https://doi.org/10.1101/2020.04.13.036079
Long T. Nguyen
aDepartment of Chemical Engineering, University of Florida, 1006 Center Drive, Gainesville, FL 32611, USA
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Brianna M. Smith
aDepartment of Chemical Engineering, University of Florida, 1006 Center Drive, Gainesville, FL 32611, USA
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Piyush K. Jain
aDepartment of Chemical Engineering, University of Florida, 1006 Center Drive, Gainesville, FL 32611, USA
bUF Health Cancer Center, University of Florida, 2033 Mowry Rd., CGRC 463, Gainesville, FL 32608, USA
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  • For correspondence: jainp@ufl.edu
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Abstract

The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3’- or 5’-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new self-catalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes.

Competing Interest Statement

The authors have declared no competing interest.

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Posted April 14, 2020.
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Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection
Long T. Nguyen, Brianna M. Smith, Piyush K. Jain
bioRxiv 2020.04.13.036079; doi: https://doi.org/10.1101/2020.04.13.036079
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Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection
Long T. Nguyen, Brianna M. Smith, Piyush K. Jain
bioRxiv 2020.04.13.036079; doi: https://doi.org/10.1101/2020.04.13.036079

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