Department of Bioengineering, University of California San Diego, USA.
Shape Therapeutics, Seattle, USA.
Methods. 2022 Sep;205:158-166. doi: 10.1016/j.ymeth.2022.06.011. Epub 2022 Jun 30.
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 both in vitro and in 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.
腺苷脱氨酶作用于 RNA（ADARs）可以通过招募它们到感兴趣的目标通过 ADAR 招募指导 RNA（adRNA）来实现特定位置的 A 到 I RNA 编辑。在本章中，我们通过两种正交策略详细介绍了实现这一目标的实验方法：一种是通过招募内源性 ADARs（即在细胞中天然表达的 ADARs）；另一种是通过招募外源性 ADARs（即递送到细胞中的 ADARs）。对于前者，我们描述了使用环形 adRNA 招募内源性 ADARs 到所需的 mRNA 靶标。这导致在体外和体内都具有强大、持久和高度转录特异性的编辑。对于后者，我们描述了使用分割 ADAR2 系统，该系统允许过表达 ADAR2 变体，可用于高度特异性地编辑腺苷，包括在具有挑战性的编辑非首选基序中的腺苷，例如那些侧翼有 5'鸟苷的腺苷。我们预计所描述的方法应该有助于跨研究和生物技术领域的 RNA 编辑应用。
