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A-to-G/C/T and C-to-T/G/A dual-function base editor for creating multi-nucleotide variants.

Bingxiu Ma1, Han Wu2, Shixue Gou3

  • 1Department of Obstetrics and Gynecology, Guangdong Provincial Key Laboratory of Major Obstetric Diseases, Guangdong Provincial Clinical Research Center for Obstetrics and Gynecology, Guangdong-Hong Kong-Macao Greater Bay Area Higher Education Joint Laboratory of Maternal-Fetal Medicine, The Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, Guangdong 510150, China.

Journal of Genetics and Genomics = Yi Chuan Xue Bao
|November 4, 2024
PubMed
Summary

We developed a dual-base editor (BDBE) to precisely install multi-nucleotide variants (MNVs), crucial for studying genetic diseases. BDBE efficiently creates nine dinucleotide variants with minimal off-target effects, advancing genetic research.

Keywords:
A-to-G/C/T and C-to-T/G/ACRISPRDual-base editorGene editingGenetic diseasesMNV

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Area of Science:

  • Molecular Biology
  • Genetic Engineering
  • Genomics

Background:

  • Multi-nucleotide variants (MNVs) are significant genetic markers implicated in numerous diseases.
  • Current technologies for the precise generation of MNVs are limited, hindering research.
  • Base editors offer a promising avenue for precise genetic modifications.

Purpose of the Study:

  • To develop a novel dual-base editor (BDBE) capable of simultaneously installing multiple types of MNVs.
  • To assess the efficiency and specificity of BDBE in human cell lines.
  • To expand the utility of base editing for comprehensive MNV research.

Main Methods:

  • Engineered a dual-base editor (BDBE) by fusing TadA-dual and engineered human N-methylpurine DNA glycosylase (eMPG) to nCas9 (D10A).
  • Tested BDBE's ability to convert adjacent CA nucleotides into all nine possible dinucleotide MNVs.
  • Evaluated BDBE4's performance in various human cell lines and compared its output to gnomAD database variants.

Main Results:

  • BDBE successfully converted A-to-B (A-to-G/C/T) and C-to-D (C-to-T/G/A) simultaneously.
  • The editor generated all nine types of dinucleotide MNVs from CA sequences with high efficiency.
  • BDBE4 demonstrated minimal off-target effects and accurately simulated all nine dinucleotide MNVs found in the gnomAD database across multiple cell lines.

Conclusions:

  • The developed dual-base editor, BDBE, significantly broadens the scope of base editing applications.
  • BDBE provides a powerful and precise tool for the creation of diverse MNVs.
  • This technology represents a valuable advancement for genetic disease research and therapeutic development.