Evaluation of DNA minicircles for delivery of adenine and cytosine base editors using activatable gene on "GO" reporter imaging systems.

Over 30,000 point mutations are associated with debilitating diseases, including many cancer types, underscoring a critical need for targeted genomic solutions. CRISPR base editors, like adenine base editors (ABEs) and cytosine base editors (CBEs), enable precise modifications by converting adenine to guanine and cytosine to thymine, respectively. Challenges in efficiency and safety concerns regarding viral vectors used in delivery limit the scope of base editing. This study introduces non-viral minicircles, bacterial-backbone-free plasmids, as a delivery vehicle for ABEs and CBEs. The research uses cells engineered with the "Gene On" (GO) reporter gene systems for tracking minicircle-delivered ABEs, CBEs, or Cas9 nickase (control), using green fluorescent protein (GFP GO ), bioluminescence reporter firefly luciferase (LUC GO ), or a highly sensitive Akaluciferase (Akaluc GO ) designed in this study. The results show that transfection of minicircles expressing CBE or ABE resulted in significantly higher GFP expression and luminescence signals over controls, with minicircles demonstrating the most substantial editing. This study presents minicircles as a new strategy for base editor delivery and develops an enhanced bioluminescence imaging reporter system for tracking ABE activity. Future studies aim to evaluate the use of minicircles in preclinical cancer models, facilitating potential clinical applications.