超过30,000个点突变与使人衰弱的疾病相关,包括许多癌症类型,强调了对靶向基因组解决方案的关键需求。CRISPR基础编辑,如腺嘌呤碱基编辑器(ABE)和胞嘧啶碱基编辑器(CBE),通过将腺嘌呤转化为鸟嘌呤和胞嘧啶转化为胸腺嘧啶来实现精确修饰,分别。关于用于递送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圆,无细菌骨架的质粒,作为ABE和CBE的交付工具。该研究使用带有“基因开启”(GO)报告基因系统的细胞来追踪微环传递的ABE,CBE,或Cas9切口酶(对照),使用绿色荧光蛋白(GFPGO),生物发光报告萤火虫荧光素酶(LUCGO),或在本研究中设计的高度敏感的Akaluciferase(AkalucGO)。结果表明,转染表达CBE或ABE的小圆导致GFP表达和发光信号明显高于对照,微型轨道展示了最实质性的编辑。这项研究提出了微圈作为基础编辑器交付的新策略,并开发了一种用于跟踪ABE活动的增强型生物发光成像报告系统。未来的研究旨在评估微圈在临床前癌症模型中的应用,促进潜在的临床应用。
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.