Ribo-On and Ribo-Off tools using a self-cleaving ribozyme allow manipulation of endogenous gene expression in C. elegans.
Ribo-On and Ribo-Off tools using a self-cleaving ribozyme allow manipulation of endogenous gene expression in C. elegans.
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DOI：
10.1038/s42003-023-05184-4
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发表时间：
2023-08-04
影响因子：
5.9
通讯作者：
Zou, Wei
中科院分区：
文献类型：
--
作者：
Fang, Jie;Wang, Jie;Wang, Yuzhi;Liu, Xiaofan;Chen, Baohui;Zou, Wei
研究基因功能有赖于对内源基因表达的有效调控。目前，只有有限的工具可用于在“开”和“关”状态之间强有力地操纵内源基因的表达。在本研究中，我们利用CRISPR/CAS9技术将T3H38核酶一个63 的编码序列插入线虫内源基因的3‘非翻译区，将内源基因的表达水平降低到几乎检测不到的水平，并产生与遗传缺失动物相似的功能丧失表型。为了实现条件性基因敲除，在内源基因的3‘非编码区插入一盒loxP侧翼的转录终止信号和核酶，通过Cre重组酶的可控表达在空间或时间上消除基因的表达。条件性内源性基因的开启可以通过注射阻断核酶自身切割活性的吗啉或使用Cre重组酶去除loxP侧翼的核酶来实现。综上所述，我们的结果表明，这些基于核酶的工具可以有效地在空间和时间上操纵内源基因的表达，并扩展了研究内源基因功能的工具包。线虫内源基因在空间和时间上的调控是通过CRISPR/Cas9将核酶序列插入到3‘非编码区，利用核酶和Cre/loxP系统的自我切割分别作为“关”和“开”的开关来完成的。
Investigating gene function relies on the efficient manipulation of endogenous gene expression. Currently, a limited number of tools are available to robustly manipulate endogenous gene expression between “on” and “off” states. In this study, we insert a 63 bp coding sequence of T3H38 ribozyme into the 3’ untranslated region (UTR) of C. elegans endogenous genes using the CRISPR/Cas9 technology, which reduces the endogenous gene expression to a nearly undetectable level and generated loss-of-function phenotypes similar to that of the genetic null animals. To achieve conditional knockout, a cassette of loxP-flanked transcriptional termination signal and ribozyme is inserted into the 3’ UTR of endogenous genes, which eliminates gene expression spatially or temporally via the controllable expression of the Cre recombinase. Conditional endogenous gene turn-on can be achieved by either injecting morpholino, which blocks the ribozyme self-cleavage activity or using the Cre recombinase to remove the loxP-flanked ribozyme. Together, our results demonstrate that these ribozyme-based tools can efficiently manipulate endogenous gene expression both in space and time and expand the toolkit for studying the functions of endogenous genes. Manipulation of endogenous genes in space and time in C. elegans is accomplished by inserting a ribozyme sequence into the 3’UTR with CRISPR/Cas9, utilizing self-cleavage of the ribozyme and Cre/loxP system as an “off and “on” switch, respectively.
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