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Highly Efficient A-to-G Editing in PFFs via Multiple ABEs.

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|April 28, 2023
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Summary

Adenine base editors (ABEs) enable precise A-to-G (T-to-C) genome editing in porcine cells without double-strand breaks. This study evaluated five ABE variants, finding dual sgRNA vectors enhanced efficiency and identified minimal DNA off-target effects.

Keywords:
ABEPFFRNA editingbase editingstart-codon mutation

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Area of Science:

  • Molecular Biology
  • Genome Editing
  • Biotechnology

Background:

  • CRISPR-mediated base editing offers precise genome modification without double-strand breaks.
  • Adenine base editors (ABEs) facilitate A-to-G (T-to-C) conversions, expanding genome editing capabilities.
  • Porcine models are crucial for biomedical research and agricultural applications.

Purpose of the Study:

  • To evaluate the efficacy and specificity of five distinct Adenine Base Editors (ABEs) in porcine fetal fibroblasts (PFFs).
  • To compare the efficiency of single versus dual sgRNA expression strategies for ABE-mediated editing.
  • To investigate potential DNA and RNA off-target effects of ABEs in porcine cells.

Main Methods:

  • Utilized five ABE variants (ABE7.10, ABEmax, NG-ABEmax, ABE8e, NG-ABE8e) for targeted A-to-G (T-to-C) conversions in five genomic loci within PFFs.
  • Employed both single and dual sgRNA expression vectors to assess editing efficiency.
  • Analyzed editing efficiency, activity windows, and off-target mutations at both DNA and RNA levels.

Main Results:

  • Observed variable editing efficiencies and activity windows across different loci and ABE variants.
  • Demonstrated superior editing efficiency when using a dual sgRNA strategy compared to separate vectors.
  • Confirmed no detectable off-target DNA mutations, but identified substantial off-target RNA events without significant pathway enrichment.
  • Showcased successful ABE-mediated start-codon mutation in APOE, leading to protein and mRNA level reduction.

Conclusions:

  • Adenine base editors are effective tools for precise A-to-G (T-to-C) point-mutation modifications in porcine cells.
  • The dual sgRNA vector strategy enhances editing efficiency in porcine systems.
  • While DNA off-target effects were negligible, RNA off-target events warrant further investigation for comprehensive safety assessment.