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Updated: Jan 23, 2026

A Fluorogenic Peptide Cleavage Assay to Screen for Proteolytic Activity: Applications for coronavirus spike protein activation
Published on: January 9, 2019
Engineered CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity for
Wenxia Zhang1, Haili Wang1, Dongzi Liu1
1State Key Laboratory of Virology and Biosafety, College of Life Sciences, Wuhan University, Wuhan, Hubei 430072, China.
None:
The CRISPR-Cas13 system exhibits potent RNA cleavage activity and has been widely utilized for RNA-targeting applications. However, its collateral cleavage of bystander RNAs limits in vivo therapeutic applications. In this study, we generated a series of LwaCas13a mutants through structure-based design and site-directed mutagenesis strategies. A triple mutant enCas13a (Q521R/E796A/E810A) was obtained with significantly enhanced target RNA cleavage activity along with only slightly increased collateral activity. To reduce the collateral activity, we optimized crRNA terminal extensions and obtained M1crRNA and M3crRNA variants that, in combination with enCas13a, maintained or reduced collateral activity while preserving enhanced targeted cleavage activity. Thus, by optimizing the Cas protein and crRNA, we have created an improved CRISPR-Cas13a system with enhanced target RNA cleavage activity and reduced collateral activity. This system demonstrated superior performance in targeting endogenous genes and antiviral applications. Mechanistic studies revealed that enhanced protein-crRNA interactions and altered complex conformations underlie the improved cleavage activity. This engineering approach provides a generalizable strategy for developing CRISPR-Cas systems with enhanced therapeutic potential.
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