A streamlined base editor engineering strategy to reduce bystander editing.

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Base editing (BE) offers a promising therapeutic strategy for genetic diseases by correcting pathogenic variants without DNA repair templates.
• Current base editors exhibit broad activity windows, limiting their therapeutic precision and increasing off-target effects.

Purpose of the Study:

• To engineer a base editor with enhanced specificity and a refined editing window for improved therapeutic applications.
• To develop a more precise and safer base editing tool by modifying the deaminase active center.

Main Methods:

• Integrated an oligonucleotide binding module into the TadA-8e deaminase active center to create the TadA-NW1 variant.
• Conjugated TadA-NW1 with Cas9 nickase and alternative PAM Cas9 variants.
• Evaluated editing efficiency, window size, and off-target activity compared to existing base editors (ABE8e).
• Tested the engineered editor (ABE-NW1) in a cystic fibrosis cell model targeting the CFTR W1282X variant.

Main Results:

• The engineered TadA-NW1 variant achieved robust A-to-G editing within a narrow four-nucleotide window, significantly smaller than the 10-bp window of ABEs.
• ABE-NW1 demonstrated substantially reduced Cas9-dependent and -independent off-target activity compared to ABE8e, while maintaining comparable on-target efficiency.
• TadA-NW1 was successfully reprogrammed for cytidine deamination and adenine transversion within a restricted window.
• ABE-NW1 efficiently and accurately corrected the CFTR W1282X variant in a cystic fibrosis cell model, outperforming existing ABEs.

Conclusions:

• Engineered base editors with refined activity windows enable more precise genome editing.
• The TadA-NW1 variant and its derivatives offer enhanced specificity and reduced off-target effects for therapeutic base editing.
• This study presents a streamlined strategy for re-engineering genome editors, accelerating the development of precise therapeutic base editing tools.