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使用 CRISPR-dCas13 系统转录特异性诱导终止密码子通读。

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

机构信息

Department of Biochemistry, Indian Institute of Science, Bengaluru, Karnataka, 560012, India.

Northwestern University Feinberg School of Medicine, Chicago, IL, USA.

出版信息

EMBO Rep. 2024 Apr;25(4):2118-2143. doi: 10.1038/s44319-024-00115-8. Epub 2024 Mar 18.

DOI:10.1038/s44319-024-00115-8
PMID:38499809
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11015002/
Abstract

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.

摘要

终止密码子通读（SCR）是指在 mRNA 上的终止密码子之后翻译继续进行的过程。在这里，我们描述了一种使用 CRISPR-dCas13 系统以转录选择性方式增强或诱导 SCR 的策略。使用特定的 guide RNA，我们将 dCas13 靶向到哺乳动物 AGO1 和 VEGFA mRNA 的典型终止密码子下游的区域，这些 mRNA 已知会发生自然 SCR。通读测定显示，dCas13-gRNA 复合物会增强这些 mRNA（外源性和内源性）的 SCR。这种效应与核糖体暂停有关，这在几个 SCR 事件中都有报道。我们的数据表明，CRISPR-dCas13 还可以在 GFP 和 TP53 mRNA 的终止密码子提前出现（PTC）处诱导 SCR。我们证明了该策略在诱导 HBB mRNA 中导致地中海贫血的 PTC 和 SPTA1 mRNA 中导致遗传性血球病的 PTC 通读方面的实用性。因此，CRISPR-dCas13 可以被编程为以转录选择性和终止密码子特异性的方式增强或诱导 SCR。

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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3337/11015002/f037321c14b1/44319_2024_115_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3337/11015002/1c0ad349488b/44319_2024_115_Fig7_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3337/11015002/5b8d0a9aaca2/44319_2024_115_Fig8_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/3337/11015002/d0e8639e0f1c/44319_2024_115_Fig9_ESM.jpg
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