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Updated: Oct 21, 2025
06:51
Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
Published on: May 6, 2020
4.1K
Cas12a target search and cleavage on force-stretched DNA
Marialucrezia Losito1,2,3, Quentin M Smith1,2, Matthew D Newton1,2
1Department of Infectious Disease, Section of Virology, Faculty of Medicine, Imperial College London, London W12 0NN, UK. david.rueda@imperial.ac.uk.
Physical Chemistry Chemical Physics : PCCP
|September 8, 2021
Summary
CRISPR-Cas12a uses 1D hopping to find DNA targets. Low force enables fast binding and cleavage, but mechanical stress slows search and impairs DNA cutting.
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- CRISPR-Cas12a is a versatile gene-editing tool.
- Understanding its mechanism is crucial for applications.
Purpose of the Study:
- Investigate CRISPR-Cas12a's DNA search and cleavage mechanism.
- Determine the role of mechanical forces in Cas12a activity.
Main Methods:
- Utilized optical tweezers to apply force to λ-DNA.
- Observed CRISPR-Cas12a interactions with DNA at the single-molecule level.
Main Results:
- CRISPR-Cas12a employs rapid 1D hopping for target localization.
- Efficient binding and cleavage occur at low forces (≤5 pN).
- A 1.8 nm conformational change limits the cleavage rate.
Conclusions:
- Mechanical forces significantly influence Cas12a dynamics.
- Low forces optimize Cas12a search and cleavage efficiency.
- Increased DNA stretching hinders Cas12a function.
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