# Engineered AcrIIA5 for optogenetic control of CRISPR‐Cas9‐based genome editing

## TL;DR

Researchers created a light-controlled CRISPR-Cas9 tool that can regulate multiple Cas9 proteins, improving gene editing precision and safety.

## Contribution

Developed two new optogenetic tools using AcrIIA5 variants with broad inhibition spectra for multiple Cas9 proteins.

## Key findings

- CASANOVA-A5 regulates SpCas9, SaCas9, NmeCas9, and St1Cas9 in a blue light-dependent manner.
- AcrIIA5-LOV9 shows optical regulation for SpCas9 using a blue light-dependent degron module.
- The study provides insights into amino acid mutations in light sensors for optimizing light-sensitive Acrs.

## Abstract

The CRISPR‐Cas9 system has been proven to be a powerful tool for gene editing in living cells and shows great potential in genetic disease treatment. Anti‐CRISPR (Acr)‐based optogenetic tools could spatiotemporally regulate the activity of CRISPR‐Cas9, thereby improving the precision and safety of gene editing. However, these tools could only regulate a certain Cas9 protein because of the high specificity of Acr used, limiting their further application. In this study, we developed a new optogenetic tool named CASANOVA‐A5 (CRISPR‐Cas9 activity switching via a novel optogenetic variant of AcrIIA5) by inserting the blue light sensor AsLOV2 into AcrIIA5 with a broad inhibition spectrum. We proved that the CASANOVA‐A5 could regulate the gene editing activity of SpCas9, SaCas9, NmeCas9, and St1Cas9 in a blue light‐dependent manner. Additionally, we engineered AcrIIA5‐LOV9 by integrating the blue light‐dependent degron module LOV9, showing obvious optical regulation for SpCas9. Together, our work demonstrates two feasible methods to engineer the Acrs to potent optogenetic tools and suggests systematic strategies for further optimization.

The bacterial‐derived CRISPR‐Cas9 system has been widely applied in life sciences due to its high programmability. To reduce the toxic side effects of this system, various spatiotemporal induction methods have been developed. Among these, the light‐sensitive induction system based on anti‐CRISPR (Acr) proteins showed high sensitivity. However, these systems were based on Acrs with narrow inhibition spectra, limiting their ability to regulate the growing number of other Cas9 proteins. This study developed a series of light‐sensitive AcrIIA5 variants that can broadly inhibit various Cas9 systems that were widely used. Furthermore, this study provides a detailed examination of the effectiveness of amino acid mutations in the light sensor, offering valuable insights for the development and optimization of other light‐sensitive Acrs.