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C-to-G editing generates double-strand breaks causing deletion, transversion and translocation.

Min Emma Huang1,2, Yining Qin2, Yafang Shang1,2

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Base editors (BEs) can cause DNA double-strand breaks (DSBs), leading to unwanted byproducts. Shielding abasic sites with HMCES reduces these DSBs, making base editing safer for therapeutic use.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • Base editors (BEs) are gene-editing tools that create precise DNA base substitutions.
  • Unclear mechanisms lead to low-frequency stochastic byproducts during base editing.
  • Understanding these byproducts is crucial for therapeutic applications of BEs.

Purpose of the Study:

  • To investigate the mechanisms behind byproduct formation in base editors.
  • To identify the role of DNA repair pathways in base editor-induced DNA damage.
  • To develop strategies for minimizing unwanted byproducts during base editing.

Main Methods:

  • In-depth outcome profiling of base editor activity.
  • Genetic dissection of DNA repair factor involvement.
  • Screening of DNA repair factors and abasic site processing.
  • Utilizing the enzyme HMCES to shield abasic sites.

Main Results:

  • C-to-G base editors (CGBEs) generate intermediate double-strand breaks (DSBs) at the editing site.
  • Imperfect DSB repair leads to deletions, insertions, and transversions.
  • Chromosomal translocations were observed between on-target and off-target sites.
  • Abasic site processing is central to DSB formation.
  • HMCES shielding significantly reduced CGBE-initiated DSBs without impacting desired substitutions.

Conclusions:

  • CGBEs can initiate deleterious intermediate DSBs, necessitating caution for therapeutic use.
  • HMCES-aided base editing presents a promising strategy for safer gene editing tools.
  • Minimizing DSB-triggered events is achievable through abasic site shielding.