Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5′-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

招募作用于 RNA 的内源性编辑酶腺苷脱氨酶 (ADAR) 和定制的引导 RNA 进行腺苷到肌苷 (A-to-I) RNA 碱基编辑，有望在 RNA 水平上安全地操纵遗传信息。然而，编辑的精度和效率往往会因旁观者脱靶编辑而受到影响。在这里，我们发现在主导旁观者编辑的 5′-U A N 三联体中，G•U 摆动​​碱基对有效地减轻了脱靶事件，同时保持了较高的目标效率。该策略普遍适用于现有的 A 到 I RNA 碱基编辑系统，并补充了其他抑制方法，例如 G•A 错配和尿苷 (U) 耗尽。将摆动碱基配对与 CLUSTER 方法的环化格式相结合，可以对细胞培养物中 Mecp2 转录本中的疾病相关突变进行高度精确和高效的编辑（高达 87%）。仅病毒介导的引导RNA递送就实现了小鼠Rett综合征模型中枢神经系统中MeCP2蛋白的功能恢复，编辑率高达19%，并且体内旁观者控制良好。