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Single-molecule assay guided crRNA optimization enhances specific microRNA detection by CRISPR-Cas12a

Sensors and Actuators B: Chemical. 2024-05; 
Kaizhao Chen, Wenjun Sun, Mingtian Zhong, Jiaqi Xie, Yongkun Huo, Xiuqi Lu, Ziting Chen, Bo Sun, Xingxu Huang, Xinjie Wang, Ming Liu , Xiaodong Ma, Peixiang Ma
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Proteins, Expression, Isolation and Analysis All peak fractions were analyzed using SurePAGE, 4–12% (GenScript). Get A Quote

摘要

CRISPR-Cas12a is a promising tool for nucleic acid detection. However, due to the protein flexibility, Cas12a tolerates mismatches, which limits its specificity. In this study, the single-molecule assay revealed that the length of crRNA regulates the association kinetics between crRNA/Cas12a complex and target DNA. Short-crRNA/Cas12 associates the target dsDNA 2-fold faster than the single-nucleotide mismatched dsDNA, whereas the long-crRNA/Cas12a binds to the fully matched and single-nucleotide mismatched DNA targets with similar rates. These findings are further corroborated by electrophoretic mobility shift assay (EMSA) and double-stranded DNA (dsDNA) cleavage results. Inspired by these findings, we establis... More

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