Transcript-specific induction of stop codon readthrough using a CRISPR-dCas13 system

Stop codon readthrough (SCR) is the process where translation continues beyond a stop codon on an mRNA. Here, we describe a strategy to enhance or induce SCR in a transcript-selective manner using a CRISPR-dCas13 system. Using specific guide RNAs, we target dCas13 to the region downstream of canonical stop codons of mammalian AGO1 and VEGFA mRNAs, known to exhibit natural SCR. Readthrough assays reveal enhanced SCR of these mRNAs (both exogenous and endogenous) caused by the dCas13-gRNA complexes. This effect is associated with ribosomal pausing, which has been reported for several SCR events. Our data show that CRISPR-dCas13 can also induce SCR across premature termination codons (PTCs) in the mRNAs of green fluorescent protein and TP53. We demonstrate the utility of this strategy in the induction of readthrough across the thalassemia-causing PTC in HBB mRNA and hereditary spherocytosis-causing PTC in SPTA1 mRNA. Thus, CRISPR-dCas13 can be programmed to enhance or induce SCR in a transcript-selective and stop codon-specific manner.终止密码子通读（Stop codon readthrough, SCR）是指翻译过程在信使RNA（messenger RNA, mRNA）的终止密码子下游继续进行的现象。本研究描述了一种利用CRISPR-dCas13系统以转录本选择性方式增强或诱导SCR的策略。通过使用特异性向导RNA（guide RNAs, gRNAs），我们将dCas13靶向至此前已被证实存在天然SCR现象的哺乳动物AGO1和VEGFA mRNA的经典终止密码子下游区域。通读测定结果显示，dCas13-gRNA复合物可增强这些mRNA（外源性及内源性）的SCR水平。该效应与核糖体暂停相关，而核糖体暂停已在多项SCR事件中被报道。我们的数据表明，CRISPR-dCas13系统还可诱导绿色荧光蛋白（green fluorescent protein, GFP）以及TP53 mRNA中提前终止密码子（premature termination codons, PTCs）介导的通读。本研究还验证了该策略在诱导HBB mRNA中导致地中海贫血的PTC以及SPTA1 mRNA中导致遗传性球形红细胞增多症的PTC实现通读方面的应用价值。综上，CRISPR-dCas13可通过编程以转录本选择性、终止密码子特异性的方式增强或诱导SCR。