CRISPR-mediated selective DNA cleavage has revolutionized gene editing, but analogous methods to manipulate RNA, thus preserving the genome and avoiding potential adverse side effects, are not available. In their paper, Nemudraia et al. present an approach for the sequence-specific deletion of transcripts in human cells that makes use of type III-A CRISPR complexes, which cleave RNA in six-nucleotide increments. Type III-A CRISPR complexes have been previously used to knockdown RNA, but as shown here, the resulting CRISPR-guided RNA breaks are repaired by RtcB ligase in human cells, allowing for programmable RNA editing, for instance to restore gene function. As a proof-of-principle, the authors use this technology to remove premature stop codons in a reporter plasmid to restore protein expression in vitro, as well as excise clinically relevant nonsense mutations in the CFTR transcript in human bronchial epithelial cells carrying a disease allele. Furthermore, as type III CRISPR systems only require complementarity between the RNA guide and the RNA target, this method is highly adaptable and not restricted to the presence of any adjacent sequence motifs. Together, this work presents a versatile means to precisely edit RNA with potential therapeutic applications.

Original reference: Science 384, 808–814 (2024)

CRISPR 介导的选择性 DNA 切割彻底改变了基因编辑，但还没有类似的方法来操纵 RNA，从而保存基因组并避免潜在的不利副作用。 Nemudraia 等人在他们的论文中。提出了一种在人类细胞中序列特异性删除转录物的方法，该方法利用 III-A 型 CRISPR 复合物，以 6 个核苷酸的增量切割 RNA。 III-A 型 CRISPR 复合物以前曾用于敲低 RNA，但如图所示，由此产生的 CRISPR 引导的 RNA 断裂可由人类细胞中的 RtcB 连接酶修复，从而允许进行可编程 RNA 编辑，例如恢复基因功能。作为原理验证，作者使用该技术去除报告质粒中的过早终止密码子，以恢复体外蛋白质表达，并切除携带疾病等位基因的人支气管上皮细胞中CFTR转录本中临床相关的无义突变。此外，由于III型CRISPR系统仅需要RNA向导和RNA靶标之间的互补性，因此该方法具有高度适应性，并且不限于任何相邻序列基序的存在。总之，这项工作提供了一种精确编辑 RNA 的通用方法，具有潜在的治疗应用。

原始参考文献： Science 384 , 808–814 (2024)