Robust and heritable knockdown of gene expression using a self-cleaving ribozyme in Drosophila.

IF 3.3 3区 生物学
Genetics Pub Date : 2024-05-03 DOI:10.1093/genetics/iyae067
Kevin G Nyberg, Fritz Gerald Navales, Eren Keles, Joseph Q Nguyen, Laura M Hertz, Richard W Carthew
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引用次数: 0

Abstract

The current toolkit for genetic manipulation in the model animal Drosophila melanogaster is extensive and versatile but not without its limitations. Here, we report a powerful and heritable method to knockdown gene expression in D. melanogaster using the self-cleaving N79 hammerhead ribozyme, a modification of a naturally occurring ribozyme found in the parasite Schistosoma mansoni. A 111 bp ribozyme cassette, consisting of the N79 ribozyme surrounded by insulating spacer sequences, was inserted into four independent long noncoding RNA genes as well as the male-specific splice variant of doublesex using scarless CRISPR/Cas9-mediated genome editing. Ribozyme-induced RNA cleavage resulted in robust destruction of 3' fragments typically exceeding 90%. Single molecule RNA fluorescence in situ hybridization results suggest that cleavage and destruction can even occur for nascent transcribing RNAs. Knockdown was highly specific to the targeted RNA, with no adverse effects observed in neighboring genes or the other splice variants. To control for potential effects produced by the simple insertion of 111 nucleotides into genes, we tested multiple catalytically inactive ribozyme variants and found that a variant with scrambled N79 sequence best recapitulated natural RNA levels. Thus, self-cleaving ribozymes offer a novel approach for powerful gene knockdown in Drosophila, with potential applications for the study of noncoding RNAs, nuclear-localized RNAs, and specific splice variants of protein-coding genes.
在果蝇中使用自裂解核糖酶稳健并可遗传地敲除基因表达。
目前对模式动物黑腹果蝇进行遗传操作的工具包既广泛又通用,但也有其局限性。在这里,我们报告了一种利用自裂解 N79 锤头核糖酶敲除黑腹果蝇基因表达的强大且可遗传的方法,N79 核糖酶是对曼氏血吸虫寄生虫中天然存在的核糖酶的改良。利用无痕 CRISPR/Cas9 介导的基因组编辑技术,将由绝缘间隔序列包围的 N79 核糖酶组成的 111 bp 核糖酶盒插入到四个独立的长非编码 RNA 基因以及双倍体的雄性特异性剪接变体中。核糖酶诱导的 RNA 裂解导致 3' 片段被强力破坏,破坏率通常超过 90%。单分子 RNA 荧光原位杂交结果表明,新生转录 RNA 甚至也会发生裂解和破坏。基因敲除对目标 RNA 具有高度特异性,对邻近基因或其他剪接变体没有不良影响。为了控制简单地在基因中插入 111 个核苷酸可能产生的影响,我们测试了多种催化活性不高的核糖酶变体,结果发现带有乱码 N79 序列的变体最能再现天然 RNA 水平。因此,自裂解核糖酶为果蝇的强效基因敲除提供了一种新方法,有望应用于研究非编码 RNA、核定位 RNA 和蛋白质编码基因的特定剪接变体。
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来源期刊
Genetics
Genetics 生物-遗传学
CiteScore
6.20
自引率
6.10%
发文量
177
期刊介绍: GENETICS is published by the Genetics Society of America, a scholarly society that seeks to deepen our understanding of the living world by advancing our understanding of genetics. Since 1916, GENETICS has published high-quality, original research presenting novel findings bearing on genetics and genomics. The journal publishes empirical studies of organisms ranging from microbes to humans, as well as theoretical work. While it has an illustrious history, GENETICS has changed along with the communities it serves: it is not your mentor''s journal. The editors make decisions quickly – in around 30 days – without sacrificing the excellence and scholarship for which the journal has long been known. GENETICS is a peer reviewed, peer-edited journal, with an international reach and increasing visibility and impact. All editorial decisions are made through collaboration of at least two editors who are practicing scientists. GENETICS is constantly innovating: expanded types of content include Reviews, Commentary (current issues of interest to geneticists), Perspectives (historical), Primers (to introduce primary literature into the classroom), Toolbox Reviews, plus YeastBook, FlyBook, and WormBook (coming spring 2016). For particularly time-sensitive results, we publish Communications. As part of our mission to serve our communities, we''ve published thematic collections, including Genomic Selection, Multiparental Populations, Mouse Collaborative Cross, and the Genetics of Sex.
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