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Updated: Nov 15, 2025
09:51
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
Published on: May 25, 2018
34.9K
Cas9 deactivation with photocleavable guide RNAs.
Roger S Zou1, Yang Liu2, Bin Wu2
1Department of Biomedical Engineering, Johns Hopkins University, Baltimore, MD 21205, USA.
Molecular Cell
|March 4, 2021
Summary
Photocleavable guide RNAs (pcRNAs) offer light-based control for CRISPR-Cas9 genome editing. This engineered approach provides rapid and complete deactivation, enhancing precision and safety in gene editing applications.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- CRISPR-Cas9 technology requires precise control for enhanced safety and broader applications.
- Current CRISPR inhibition methods face challenges including separate inhibitor delivery, incomplete deactivation, and slow kinetics.
Purpose of the Study:
- To engineer photocleavable guide RNAs (pcRNAs) for light-inducible deactivation of CRISPR-Cas9 systems.
- To overcome limitations of existing CRISPR inhibition strategies.
Main Methods:
- Development of photocleavable guide RNAs (pcRNAs) integrated with Cas9 nucleases and base editors.
- Evaluation of pcRNA-mediated deactivation speed, completeness, and specificity.
- Time-resolved analysis of Cas9 and base editor activity duration for efficient editing.
Main Results:
- pcRNA enabled the fastest (<1 minute) and most complete (<1% residual indels) CRISPR-Cas9 deactivation.
- Significantly enhanced specificity was observed with wild-type Cas9.
- Defined time windows (12-36h for Cas9, 2-4h for base editors) achieved high editing efficiency.
Conclusions:
- pcRNA provides a built-in, light-based deactivation mechanism for CRISPR-Cas9 and base editors.
- This technology accelerates and improves the completeness of CRISPR deactivation.
- pcRNA is valuable for studying cellular responses to DNA damage and advancing precision genome editing.
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