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Engineered Sdd7 cytosine base editors with enhanced specificity.

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Area of Science:

  • Molecular Biology
  • Genome Editing Technologies

Background:

  • Cytosine base editors (CBEs) enable precise C-to-T conversions without double-strand breaks.
  • The Sdd7 cytosine deaminase shows high activity but causes off-target mutations, including bystander edits upstream and within the protospacer.

Purpose of the Study:

  • To engineer Sdd7 variants with enhanced specificity and reduced off-target activity.
  • To evaluate the efficacy of Sdd7 variants delivered via engineered virus-like particles (eVLPs).

Main Methods:

  • Engineering of two Sdd7 variants (Sdd7e1 and Sdd7e2).
  • Assessment of on-target efficiency and off-target effects, including bystander mutations.
  • Delivery of base editors as ribonucleoproteins via eVLPs.

Main Results:

  • Sdd7e1 and Sdd7e2 variants demonstrated reduced bystander editing and narrower editing windows compared to wild-type Sdd7.
  • Off-target activity was significantly lowered in the engineered variants.
  • eVLP delivery nearly eliminated bystander edits and increased precise single-point mutations.

Conclusions:

  • Sdd7e1 and Sdd7e2 represent high-fidelity CBEs with improved specificity.
  • eVLP delivery further enhances the safety and precision of these CBEs.
  • These engineered CBEs are promising for therapeutic genome editing applications.