Methods for recruiting endogenous and exogenous ADAR enzymes for site-specific RNA editing.
Methods for recruiting endogenous and exogenous ADAR enzymes for site-specific RNA editing.
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招募内源性和外源性 ADAR 酶进行位点特异性 RNA 编辑的方法。
DOI：
10.1016/j.ymeth.2022.06.011
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发表时间：
2022
期刊：
影响因子：
--
通讯作者：
Mali,Prashant
中科院分区：
文献类型：
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作者：
Xiang,Yichen;Katrekar,Dhruva;Mali,Prashant
作用于RNA的腺苷脱氨酶（ADAR）可以通过经由ADAR募集向导RNA（adRNA）将它们募集到感兴趣的靶标来重新利用以实现位点特异性A至I RNA编辑。在本章中，我们详细介绍了通过两种正交策略实现这一目标的实验方法：一种是通过募集内源性ADAR（即已经在细胞中天然表达的ADAR）;另一种是通过募集外源性ADAR（即递送到细胞中的ADAR）。对于前者，我们描述了使用环状adRNA将内源性ADAR募集到所需的mRNA靶标。这导致在体外和体内进行稳健、持续和高度转录特异性的编辑。对于后者，我们描述了分裂ADAR 2系统的使用，该系统允许ADAR 2变体的过表达，这些变体可用于以高特异性编辑腺苷，包括挑战编辑非优选基序中的腺苷，例如侧翼为5′鸟苷的那些。我们预计所描述的方法应该有助于跨研究和生物技术环境的RNA编辑应用。
Adenosine deaminases acting on RNA (ADARs) can be repurposed to achieve site-specific A-to-I RNA editing by recruiting them to a target of interest via an ADAR-recruiting guide RNA (adRNA). In this chapter, we present details towards experimental methods to enable this via two orthogonal strategies: one, via recruitment of endogenous ADARs (i.e. ADARs already natively expressed in cells); and two, via recruitment of exogenous ADARs (i.e. ADARs delivered into cells). Towards the former, we describe the use of circular adRNAs to recruit endogenous ADARs to a desired mRNA target. This results in robust, persistent and highly transcript specific editing bothin vitroandin vivo. Towards the latter, we describe the use of a split-ADAR2 system, which allows for overexpression of ADAR2 variants that can be utilized to edit adenosines with high specificity, including at challenging to edit adenosines in non-preferred motifs such as those flanked by a 5′ guanosine. We anticipate the described methods should facilitate RNA editing applications across research and biotechnology settings.