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Single molecule methods for studying CRISPR Cas9-induced DNA unwinding.
Ikenna C Okafor1, Janice Choi2, Taekjip Ha3
1Department of Biology, Johns Hopkins University, Baltimore, MD, USA.
Methods (San Diego, Calif.)
|November 12, 2021
Summary
CRISPR Cas9 DNA unwinding does not require ATP, unlike helicases. Researchers used single-molecule FRET to study how guide RNA mismatches affect DNA unwinding and Cas9 nuclease activity.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- CRISPR-Cas9 is a powerful genome editing tool.
- CRISPR proteins like Cas9 and Cas12a unwind DNA without ATP, unlike helicases.
- They utilize binding energy and guide RNA basepairing for DNA unwinding.
Purpose of the Study:
- To investigate the mechanism of DNA unwinding by CRISPR-Cas9.
- To explore the role of guide RNA-DNA mismatches in Cas9 activity.
- To probe the conformational changes of Cas9 during DNA cleavage.
Main Methods:
- Single-molecule Förster Resonance Energy Transfer (smFRET) was employed.
- smFRET was used to monitor DNA unwinding dynamics.
- The positioning of the Cas9 HNH nuclease domain was tracked.
Main Results:
- DNA unwinding by CRISPR-Cas9 is driven by binding energy and guide RNA complementarity.
- Mismatches between guide RNA and the target DNA sequence influence unwinding.
- smFRET successfully probed Cas9 conformational states and nuclease domain positioning.
Conclusions:
- CRISPR-Cas9 utilizes a unique ATP-independent DNA unwinding mechanism.
- Guide RNA accuracy is critical for Cas9-mediated DNA cleavage.
- smFRET is a valuable technique for studying CRISPR-Cas9 mechanisms at the single-molecule level.
