Clustered regularly interspaced short palindromic repeats–Cas13 effectors are used for RNA editing but the adeno-associated virus (AAV) packaging limitations because of their big sizes hinder their therapeutic application. Here we report the identification of the Cas13j family, with LepCas13j (529 aa) and ChiCas13j (424 aa) being the smallest and most highly efficient variants for RNA interference. The miniaturized Cas13j proteins enable the development of compact RNA base editors. Chi-RESCUE-S, by fusing dChiCas13j with hADAR2dd, demonstrates high efficiency and specificity in A-to-G and C-to-U conversions. Importantly, this system is compatible with single-AAV packaging without the need for protein sequence truncation. It successfully corrected pathogenic mutations, such as APOC3^D65N and SCN9A^R896Q, to the wild-type forms. In addition, we developed an optimized system, Chi-RESCUE-S-mini3, which pioneered efficient in vivo C-to-U RNA editing of PCSK9 in mice through single-AAV delivery, resulting in reduced total cholesterol levels. These results highlight the potential of Cas13j to treat human diseases.

成簇的规则间隔的短回文重复序列——Cas13效应子用于RNA编辑，但腺相关病毒（AAV）由于尺寸较大而受到包装限制，阻碍了其治疗应用。在这里，我们报告了 Cas13j 家族的鉴定，其中 LepCas13j (529 个氨基酸) 和 ChiCas13j (424 个氨基酸) 是最小且最高效的 RNA 干扰变体。小型化的 Cas13j 蛋白能够开发紧凑的 RNA 碱基编辑器。 Chi-RESCUE-S 通过将 dChiCas13j 与 hADAR2dd 融合，在 A-to-G 和 C-to-U 转换中表现出高效率和特异性。重要的是，该系统与单 AAV 包装兼容，无需蛋白质序列截断。它成功地将APOC3 ^D65N和SCN9A ^R896Q等致病突变纠正为野生型形式。此外，我们开发了一种优化系统 Chi-RESCUE-S-mini3，该系统率先通过单 AAV 递送对小鼠体内PCSK9进行有效的体内 C-to-U RNA 编辑，从而降低总胆固醇水平。这些结果凸显了 Cas13j 治疗人类疾病的潜力。