Efficient genome editing with Cas9-sgRNA in vivo has required the use of viral delivery systems, which have limitations for clinical applications. Translational efforts to develop other RNA therapeutics have shown that judicious chemical modification of RNAs can improve therapeutic efficacy by reducing susceptibility to nuclease degradation. Guided by the structure of the Cas9-sgRNA complex, we identify regions of sgRNA that can be modified while maintaining or enhancing genome-editing activity, and we develop an optimal set of chemical modifications for in vivo applications. Using lipid nanoparticle formulations of these enhanced sgRNAs (e-sgRNA) and mRNA encoding Cas9, we show that a single intravenous injection into mice induces >80% editing of Pcsk9 in the liver. Serum Pcsk9 is reduced to undetectable levels, and cholesterol levels are significantly lowered about 35% to 40% in animals. This strategy may enable non-viral, Cas9-based genome editing in the liver in clinical settings.

中文翻译：

---

对引导RNA进行结构引导的化学修饰可实现有效的非病毒体内基因组编辑。


在体内使用 Cas9-sgRNA 进行有效的基因组编辑需要使用病毒递送系统，这对临床应用有限制。开发其他 RNA 疗法的转化努力表明，明智的 RNA 化学修饰可以通过降低对核酸酶降解的敏感性来提高治疗效果。在 Cas9-sgRNA 复合物结构的指导下，我们确定了可以在维持或增强基因组编辑活性的同时进行修饰的 sgRNA 区域，并开发了一组用于体内应用的最佳化学修饰。使用这些增强的 sgRNA (e-sgRNA) 和编码 Cas9 的 mRNA 的脂质纳米颗粒制剂，我们发现单次静脉注射到小鼠体内可诱导肝脏中 Pcsk9 的编辑超过 80%。动物体内血清 Pcsk9 降低至检测不到的水平，胆固醇水平显着降低约 35% 至 40%。该策略可能在临床环境中实现肝脏中基于 Cas9 的非病毒基因组编辑。