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Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • Miniature CRISPR-Cas12f systems offer potential for gene editing but their base editor derivatives (miniCBEs and miniABEs) require further investigation.
  • Functional miniature cytosine base editors (miniCBEs) have not been established, limiting their application.

Purpose of the Study:

  • To develop and characterize novel Cas12f-derived miniature base editors with enhanced editing activity and broader targeting capabilities.
  • To engineer highly precise miniCBEs with minimized off-target effects by improving deaminase components.

Main Methods:

  • Generation of various Cas12f-derived miniCBEs and miniABEs.
  • Engineering of TadA deaminase through mutagenesis screening for improved precision.
  • Assessment of editing efficacy, targeting range, and off-target effects in vitro.
  • In vivo delivery of miniCBEs and miniABEs via adeno-associated virus in the brain.

Main Results:

  • Cas12f-derived miniCBEs and miniABEs demonstrated improved editing activities and expanded targeting scopes.
  • Traditional cytidine deaminases in miniCBEs resulted in wide editing windows but significant off-target effects.
  • Engineered TadA deaminase yielded potent miniCBEs with high precision and reduced off-target mutations.
  • Newly designed miniCBEs and miniABEs successfully corrected pathogenic mutations in cell lines and induced genetic mutations in vivo.

Conclusions:

  • This study presents novel strategies for developing cytosine base editors (CBEs) and expands the toolkit of miniature base editors.
  • The developed miniCBEs and miniABEs offer promising therapeutic potential for clinical applications in gene therapy.