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Related Experiment Video

Updated: Jan 13, 2026

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

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

Haonan Zhang^1,2, Ying Yang¹, Tianxiang Yang²

¹Central Hospital of Dalian University of Technology, Xinan Road No. 826, Dalian 116024, China.

|October 29, 2025

Summary

This summary is machine-generated.

Scientists engineered MAD7_HF, a high-fidelity CRISPR-Cas12a variant, using a novel bacterial screening system. This advanced genome editing tool significantly reduces off-target cleavage while maintaining high on-target efficiency.

Keywords:

MAD7_HF bacterial screening system high specificity off-target

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CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.

Published on: December 11, 2020

Area of Science:

Molecular Biology
Genetics
Biotechnology

Background:

CRISPR-Cas nucleases offer precise genome editing capabilities.
Off-target cleavage is a significant limitation in current CRISPR-Cas technologies.
Developing highly specific genome editing tools is crucial for therapeutic applications.

Purpose of the Study:

To engineer a high-fidelity variant of the MAD7 nuclease with improved specificity.
To develop and validate a high-throughput bacterial screening system for identifying precise genome editing tools.
To characterize the performance of the engineered MAD7_HF variant in reducing off-target activity.

Main Methods:

Engineered the MAD7 nuclease using a bacterial screening system based on the toxic gene ccdB.
Employed iterative selection and sequencing to identify high-fidelity variants.
Validated the specificity and efficiency of the engineered variant (MAD7_HF) in Escherichia coli assays.

Main Results:

Identified MAD7_HF with three key substitutions (R187C, S350T, K1019N).
MAD7_HF demonstrated over a 20-fold reduction in off-target cleavage compared to wild-type MAD7.
On-target editing efficiency of MAD7_HF remained comparable to wild-type MAD7.

Conclusions:

The developed bacterial screening strategy is effective for identifying Cas12a variants with enhanced specificity.
MAD7_HF represents a significant advancement in precision genome editing by minimizing off-target effects.
This work provides a framework for developing next-generation, highly specific genome editing tools.