# Engineering a High-Fidelity MAD7 Variant with Enhanced Specificity for Precision Genome Editing via CcdB-Based Bacterial Screening

## TL;DR

Scientists engineered a more precise CRISPR nuclease variant, MAD7_HF, using a bacterial screening system to reduce off-target effects in genome editing.

## Contribution

A novel bacterial screening system using CcdB was developed to identify a high-fidelity MAD7 variant with enhanced specificity.

## Key findings

- MAD7_HF showed a >20-fold reduction in off-target cleavage in E. coli.
- Three mutations (R187C, S350T, K1019N) improved on-target discrimination.
- Structural modeling linked mutations to stabilized guide RNA-DNA hybrid and reduced mismatch interactions.

## Abstract

CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) nucleases enable precise genome editing, but off-target cleavage remains a critical challenge. Here, we report the development of MAD7_HF, a high-fidelity variant of the MAD7 nuclease engineered through a bacterial screening system leveraging the DNA gyrase-targeting toxic gene ccdB. This system couples survival to efficient on-target cleavage and minimal off-target activity, mimicking the transient action required for high-precision editing. Through iterative selection and sequencing validation, we identified MAD7_HF, harboring three substitutions (R187C, S350T, K1019N) that enhanced discrimination between on- and off-target sites. In Escherichia coli assays, MAD7_HF exhibited a >20-fold reduction in off-target cleavage across multiple mismatch contexts while maintaining on-target efficiency comparable to wild-type MAD7. Structural modeling revealed that these mutations stabilize the guide RNA-DNA hybrid at on-target sites and weaken interactions with mismatched sequences. This work establishes a high-throughput bacterial screening strategy that allows the identification of Cas12a variants with improved specificity at a given target site, providing a useful framework for future efforts to develop precision genome-editing tools.