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Updated: Jul 11, 2025

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
Published on: October 18, 2022
Rapid characterization of anti-CRISPR proteins and optogenetically engineered variants using a versatile plasmid
Guoxu Song1,2, Chunhong Tian1,2,3, Jiahui Li1,2,3
1Key Laboratory of Epigenetic Regulation and Intervention, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China.
Researchers developed a new system to quickly find anti-CRISPR (Acr) proteins and create controllable CRISPR tools. This system led to the discovery of new Acrs and light-activated Cas9 control systems for genome editing.
Area of Science:
- Molecular Biology
- Biotechnology
- Genetics
Background:
- Anti-CRISPR (Acr) proteins inhibit CRISPR-Cas systems, offering potential for precise gene editing control.
- Characterizing novel Acrs and developing Acr-based technologies is currently time-consuming.
Purpose of the Study:
- To establish a rapid screening system for novel anti-CRISPR proteins.
- To develop controllable Acr-based tools for CRISPR-Cas applications.
Main Methods:
- Developed a versatile plasmid interference with CRISPR interference (PICI) system in E. coli.
- Discovered and characterized novel type II-A Acrs (AcrIIA33 and AcrIIA34).
- Engineered optogenetically controlled AcrIIA4 variants (OPERA4) using light-oxygen-voltage 2 (LOV2) domains.
Main Results:
- Identified two novel type II-A Acrs, AcrIIA33 and AcrIIA34, inhibiting SpyCas9 DNA binding.
- Created robust, light-inducible OPERA4 variants with ~1000-fold control over SpyCas9 activity.
- Demonstrated light-controllable genome editing in both prokaryotic and human cells.
Conclusions:
- The PICI system enables rapid characterization of anti-CRISPR proteins.
- OPERA4 variants provide robust, light-dependent control for CRISPR-Cas9 applications.
- This work advances the development of controllable tools for CRISPR-based technologies.
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