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

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.