Programmable RNA pseudouridylation has emerged as a new type of RNA base editor to suppress premature termination codons (PTCs) that can lead to truncated and nonfunctional proteins. However, current methods to correct disease-associated PTCs suffer from low efficiency and limited precision. Here we develop RESTART v3, which uses near-cognate tRNAs to improve the readthrough efficiency of pseudouridine-modified PTCs. We show an average of ~5-fold (range: 2.1- to 9.5-fold) higher editing efficiency than RESTART v2 in cultured cells and achieve functional PTC readthrough in disease cell models of cystic fibrosis and Hurler syndrome. Furthermore, RESTART v3 enables accurate incorporation of the original amino acid for nearly half of the PTC sites, considering the naturally occurring frequencies of sense-to-nonsense codons, without affecting normal termination codons. Although off-target sites were detected, we did not observe changes to the coding information or the expression level of transcripts, and the overall natural tRNA abundance remained constant.

可编程 RNA 假尿苷化已成为一种新型的 RNA 碱基编辑器，用于抑制可导致蛋白质截短和无功能蛋白质的过早终止密码子 （PTC）。然而，目前纠正疾病相关 PTC 的方法效率低且精度有限。在这里，我们开发了 RESTART v3，它使用近同源 tRNA 来提高假尿苷修饰的 PTC 的通读效率。我们显示，在培养细胞中，编辑效率平均比 RESTART v2 高 ~5 倍（范围：2.1 至 9.5 倍），并在囊性纤维化和 Hurler 综合征的疾病细胞模型中实现功能性 PTC 通读。此外，考虑到正义到无义密码子的天然发生频率，RESTART v3 能够准确掺入近一半的 PTC 位点的原始氨基酸，而不会影响正常的终止密码子。尽管检测到脱靶位点，但我们没有观察到编码信息或转录本表达水平的变化，并且总体天然 tRNA 丰度保持不变。