Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection.
Nat Commun
; 11(1): 4906, 2020 09 30.
Article
in English
| MEDLINE | ID: covidwho-807811
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.
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Bacterial Proteins
/
RNA, Viral
/
Trans-Activators
/
Nucleic Acid Amplification Techniques
/
Endodeoxyribonucleases
/
CRISPR-Associated Proteins
/
Betacoronavirus
Type of study:
Diagnostic study
/
Prognostic study
Language:
English
Journal:
Nat Commun
Journal subject:
Biology
/
Science
Year:
2020
Document Type:
Article
Affiliation country:
S41467-020-18615-1
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