Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids.
Cell Rep Med
; 4(5): 101037, 2023 05 16.
Article
in English
| MEDLINE | ID: covidwho-2317671
ABSTRACT
CRISPR-Cas-based diagnostics have the potential to elevate nucleic acid detection. CRISPR-Cas systems can be combined with a pre-amplification step in a one-pot reaction to simplify the workflow and reduce carryover contamination. Here, we report an engineered Cas12b with improved thermostability that falls within the optimal temperature range (60°C-65°C) of reverse transcription-loop-mediated isothermal amplification (RT-LAMP). Using de novo structural analyses, we introduce mutations to wild-type BrCas12b to tighten its hydrophobic cores, thereby enhancing thermostability. The one-pot detection assay utilizing the engineered BrCas12b, called SPLENDID (single-pot LAMP-mediated engineered BrCas12b for nucleic acid detection of infectious diseases), exhibits robust trans-cleavage activity up to 67°C in a one-pot setting. We validate SPLENDID clinically in 80 serum samples for hepatitis C virus (HCV) and 66 saliva samples for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with high specificity and accuracy. We obtain results in as little as 20 min, and with the extraction process, the entire assay can be performed within an hour.
Keywords
Full text:
Available
Collection:
International databases
Database:
MEDLINE
Main subject:
Nucleic Acids
/
COVID-19
Type of study:
Diagnostic study
/
Prognostic study
Limits:
Humans
Language:
English
Journal:
Cell Rep Med
Year:
2023
Document Type:
Article
Affiliation country:
J.xcrm.2023.101037
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