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

Long T. Nguyen, Brianna M. Smith, Piyush K. Jain

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Received Date: 22nd April 20

The CRISPR/Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA but are limited to picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically-relevant nucleic acid targets, including prostate cancer, HIV, HCV, and SARS-CoV-2, with unprecedented sensitivity, achieving a limit of detection 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. By combining an isothermal amplification step with a paper-based lateral flow test, the modified crRNAs could detect SARS-CoV-2 RNA with up to 23-fold higher sensitivity within 40-60 minutes. 

Read in full at bioRxiv.

This is an abstract of a preprint hosted on an independent third party site. It has not been peer reviewed but is currently under consideration at Nature Communications.

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