(556e) Engineered CRISPR/Cas12a System As a Sensitive Diagnostic Tool for Detecting HCV, HIV, and SARS-CoV-2

CRISPR/Cas (Cluster of Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein) systems have emerged not only as a precise gene-editing tool but also as a new sensitive diagnostic tool due to additional functionality of some newly discovered Cas proteins. Class II effector proteins, such as Cas12a, are considered as a better alternative to the CRISPR/Cas9 system due to their compact size, specificity, and efficiency. Besides executing successful gene editing, once Cas12a forms a ternary complex with guide RNA (crRNA) and target dsDNA and executes a double-stranded break (referred to as cis-cleavage), it executes nonspecific cleavage of ssDNA. This attribute, known as trans-cleavage, is only activated once bound to a target that has complementary base-pairing to the guide crRNA. When combined with a FRET-based reporter, a fluorophore connected to a quencher via a short oligonucleotide, the presence of the target activator can be validated. Such a phenomenon has been efficiently harnessed by SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) to reliably detect nucleic acids.

Though rising as a versatile diagnostic tool that can detect nucleic acids with a high level of precision and accuracy based on complementary base pairing principle, current CRISPR/Cas12a systems are limited to a nanomolar detection limit without an amplification step. We, therefore, sought to overcome this limitation by finding ways to engineer CRISPR/Cas systems to enhance their sensitivity for nucleic acid detection. We extended the 3’- or 5’-ends of the crRNA with different lengths of DNA, RNA, and phosphorothioate DNA. Through selective engineering and design, we achieved amplified Cas12a trans-cleavage activity as high as 3.5-fold with our modified crRNA compared to the wild type crRNA on the target dsDNA eGFP fragment. Employing this phenomenon, we optimized a fluorescence-based detection assay that enabled us to detect nucleic acids without an amplification step with unprecedented sensitivity and with a limit of detection in femtomolar scale. This engineered crRNA-Cas12a was successfully applied towards detecting clinically relevant targets such as HCV, HIV, and SARS-CoV-2.

When combined with an isothermal target pre-amplification step such as RPA/LAMP, we observed an enhance in sensitivity down to a few copies of nucleic acids with our engineered crRNA-Cas12a system. Moreover, we combined this platform with lateral flow assay to create a rapid, portable, and inexpensive point-of-care paper strip tests that can be used by nonprofessionals. As a proof of concept, the paper strip test successfully displayed visibly positive signal when targeting synthetic RNA of SARS-CoV-2 down to 3 copies. Furthermore, such engineered point-of-care diagnostics could be extended to various other infectious diseases.