Engineered ADARs enable precision A-to-G base editing of DNA

Area of Science:

• Molecular Biology
• Gene Editing Technologies
• Biochemistry

Background:

• Adenine base editors (ABEs) facilitate A•T-to-G•C conversions, showing therapeutic promise.
• Conventional ABEs exhibit bystander nucleotide conversions, limiting precise genome editing.

Purpose of the Study:

• To develop a single-nucleotide resolution ABE (snuABE) for highly specific A-to-G base editing.
• To engineer an improved deaminase for enhanced ABE activity and specificity.

Main Methods:

• Constructed snuABE by fusing nickase Cas9 (nCas9-H840A) with the ADAR deaminase domain.
• Utilized a target-adenine guide RNA (tagRNA) to direct editing.
• Employed in silico protein evolution (EvolvePro) to engineer ADAR and 3'-end protected tagRNA.

Main Results:

• Achieved median A-to-G editing efficiency of 5.4% and maximum efficiency of 50.0% across 32 targets in HEK293T cells.
• Demonstrated no detectable DNA off-target editing at predicted off-target or R-loop sites.
• ADAR deaminase domain acts on DNA:RNA hybrids, unlike TadA in conventional ABEs.

Conclusions:

• snuABE provides a precise and safe base-editing technology by minimizing bystander conversions.
• Engineered ADAR and tagRNA significantly enhanced snuABE activity and specificity.
• snuABE holds potential for precise therapeutic genome editing applications.