Terminal Chemical Modifications of crRNAs Enable Improvement in the Performance of CRISPR-Cas for Point-of-Care Nucleic Acid Detection

CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in low-resource settings. Here, we...

Full description

Saved in:
Bibliographic Details
Published in:Analytical chemistry (Washington) 2024-10, Vol.96 (41), p.16346-16354
Main Authors: Wan, Yunzhu, Li, Sheng, Xu, Wenfei, Wang, Ke, Guo, Wenlong, Yang, Chongguang, Li, Xuhui, Zhou, Jianhua, Wang, Jiasi
Format: Article
Language:English
Subjects:
Assaying
Chemical activity
Chemical modification
CRISPR
Degradation
Environmental degradation
Nucleic acids
Performance enhancement
Portable equipment
Recombinase
Workflow
Citations: Items that this one cites
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary:CRISPR-Cas systems, harnessing their precise nucleic acid recognition via CRISPR RNA (crRNA), offer promise for the accurate testing of nucleic acids in the field. However, the inherent susceptibility of crRNA to degradation poses challenges for accurate detection in low-resource settings. Here, we utilized the chemically modified crRNA for the CRISPR-Cas-based assay (CM-CRISPR). We found that the extension and chemical modification to crRNA significantly enhanced the trans-cleavage activity of LbCas12a. The chemically modified crRNA was resistant to degradation, and CM-CRISPR showed superior detection capability in complex environments. CM-CRISPR could be combined with recombinase polymerase amplification (RPA) and applied in a droplet digital platform, enabling attomolar-level sensitivity. We also developed a portable and automated device for a digital CRISPR assay, which is amenable to point-of-care testing (POCT). The extraction-free procedure was integrated with this assay to streamline the workflow, and clinical samples were successfully detected. This work finds a simple and efficient way to improve the performance of CRISPR-Cas and develops a portable platform for POCT, representing a significant advance toward practical applications of CRISPR-based diagnostics.
ISSN:0003-2700
1520-6882
1520-6882
DOI:10.1021/acs.analchem.4c03698