超过 30,000 个点突变与衰弱性疾病（包括许多癌症类型）相关，这凸显了对靶向基因组解决方案的迫切需求。 CRISPR 碱基编辑器，如腺嘌呤碱基编辑器 (ABE) 和胞嘧啶碱基编辑器 (CBE)，分别通过将腺嘌呤转换为鸟嘌呤和将胞嘧啶转换为胸腺嘧啶来实现精确修饰。用于传递的病毒载体的效率和安全性方面的挑战限制了碱基编辑的范围。这项研究引入了非病毒小环、无细菌主链质粒作为 ABE 和 CBE 的递送载体。该研究使用经过“Gene On”（GO）报告基因系统改造的细胞，使用绿色荧光蛋白（GFPGO）、生物发光报告萤火虫荧光素酶（LUCGO）或本研究中设计了一种高度敏感的 Akaluciferase (AkalucGO)。结果表明，表达 CBE 或 ABE 的小环的转染导致 GFP 表达和发光信号显着高于对照，其中小环表现出最实质性的编辑。这项研究将小环作为碱基编辑器传递的新策略，并开发了一种用于跟踪 ABE 活性的增强型生物发光成像报告系统。未来的研究旨在评估小环在临床前癌症模型中的使用，促进潜在的临床应用。© 2024 作者。由爱思唯尔公司代表美国基因与细胞治疗学会出版。

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 (GFPGO), bioluminescence reporter firefly luciferase (LUCGO), or a highly sensitive Akaluciferase (AkalucGO) 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.© 2024 The Authors. Published by Elsevier Inc. on behalf of The American Society of Gene and Cell Therapy.