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

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.