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Mismatch Repair

Mismatch Repair

Organisms are capable of detecting and fixing nucleotide mismatches that occur during DNA replication. This sophisticated process requires identifying the new strand and replacing the erroneous bases with correct nucleotides. Mismatch repair is coordinated by many proteins in both prokaryotes and eukaryotes.
The Mutator Protein Family Plays a Key Role in DNA Mismatch Repair
The human genome has more than 3 billion base pairs of DNA per cell. Prior to cell division, that vast amount of genetic...

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Updated: Jun 29, 2025

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter

Published on: March 31, 2022

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Yaokang Wu^1,2, Yang Li^1,2, Yanfeng Liu^1,2

¹Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China.

Nucleic Acids Research

|April 3, 2024

Summary

This summary is machine-generated.

Researchers developed MultiduBE, a novel base editor enabling simultaneous genetic mutations across multiple sites. This tool accelerates the study of complex biological functions and enhances metabolic engineering in bacteria like E. coli and B. subtilis.

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Published on: December 11, 2020

Area of Science:

Synthetic biology
Molecular biology
Genetics

Background:

Understanding genetic diversity through mutagenesis is crucial for biological research and engineering.
Current methods for combinatorial in-situ mutagenesis are limited, hindering the study of complex biological functions.

Purpose of the Study:

To design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor for combinatorial in-situ mutagenesis.
To demonstrate the efficacy of MultiduBE in reprogramming cell physiology and metabolic regulation in bacteria.

Main Methods:

Development of MultiduBE using dCas12a, synthetic effectors (duBE-1a, duBE-2b) with deaminase activities, and a synthetic separator (Sp4).
Application of MultiduBE for multiplexed in-situ mutagenesis in Escherichia coli and Bacillus subtilis.
Identification of a streptomycin resistance mutation and combinatorial mutagenesis for enhanced antibiotic resistance.

Main Results:

Successful implementation of multiplexed in-situ mutagenesis in E. coli and B. subtilis.
Identification of a novel streptomycin resistance mutation in B. subtilis.
Combinatorial mutants showed significant improvements: 42% increase in surfactin and 15-fold increase in riboflavin titers compared to single-mutation controls.

Conclusions:

MultiduBE offers a convenient and efficient platform for performing multiplexed in-situ mutagenesis.
This technology facilitates complex genetic studies and metabolic engineering applications.
The tool enables rapid generation of bacterial strains with desirable traits, such as enhanced antibiotic resistance and improved metabolite production.