Abstract
The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Here, we develop a platform with engineered crRNAs and optimized conditions that enabled us to detect various clinically relevant nucleic acid targets with higher sensitivity, achieving a limit of detection in the 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 discover a 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 and with significant improvement in specificity for target recognition. Particularly, the 7-mer DNA extension to crRNA is determined to be universal and spacer-independent for enhancing the sensitivity and specificity of LbCas12a-mediated nucleic acid detection. We perform a detailed characterization of our engineered ENHANCE system with various crRNA modifications, target types, reporters, and divalent cations. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs are incorporated in a paper-based lateral flow assay that can detect the target with up to 23-fold higher sensitivity within 40–60 min.
Highlights
The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step
By combining an isothermal amplification step, this system shows improved detection of SARS-CoV-2 genomic RNA using a fluorescence-based and a paper-based lateral flow assay compared to wild-type CRISPR-Cas12a system
We discovered that ssDNA and ssRNA extensions on the 3′-end of crGFP markedly enhanced the trans-cleavage ability of targetactivated LbCas12a
Summary
The CRISPR-Cas12a RNA-guided complexes have tremendous potential for nucleic acid detection but are limited to the picomolar detection limit without an amplification step. Class 2 CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) systems, such as Cas12a (previously referred as Cpf[1], subtype V-A) and Cas13a (previously referred C2c2, subtype VI), are capable of nonspecific cleavage of ssDNA (single-stranded DNA) and RNA, respectively, in addition to successful gene editing[1,2,3] This attribute, known as trans-cleavage, is only activated once bound to an activator (ssDNA or dsDNA) 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 sequence, the presence of the target activator can be confirmed This 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[1,4,5,6,7,8]. By combining an isothermal amplification step, this system shows improved detection of SARS-CoV-2 genomic RNA using a fluorescence-based and a paper-based lateral flow assay compared to wild-type CRISPR-Cas12a system
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
