Engineering Nme2Cas9 Adenine Base Editors with Improved Activity and Targeting Scope

Area of Science:

• Molecular Biology
• Genetics
• Biotechnology

Background:

• Nme2Cas9 is a compact genome editing platform known for its accuracy and broad targeting capabilities, including adenine base editing.
• Existing Nme2Cas9 adenine base editors (Nme2-ABE) have limitations in activity and targeting scope.

Approach:

• Engineered Nme2Cas9 by inserting domains to reposition the deaminase domain closer to the target DNA strand.
• Modified the PAM-interacting domain of Nme2Cas9 by swapping it with that of SmuCas9 to broaden the targeting range.
• Utilized these enhanced Nme2Cas9 variants to correct MECP2 mutations associated with Rett syndrome.

Key Points:

• Domain-inlaid Nme2Cas9 variants demonstrated shifted editing windows and superior activity compared to N-terminally fused Nme2-ABE.
• Swapping the PAM-interacting domain expanded the editing scope to include a single-cytidine PAM.
• The enhanced editors corrected two common MECP2 mutations with minimal bystander editing.
• Validated domain-inlaid Nme2-ABEs for efficient single-AAV delivery in vivo.

Conclusions:

• Engineered Nme2Cas9 base editors exhibit increased activity and targeting scope.
• These advancements enable precise correction of disease-associated mutations like those in MECP2.
• The enhanced editors are suitable for single-AAV delivery, paving the way for in vivo gene therapy applications.