Methods for recruiting endogenous and exogenous ADAR enzymes for site-specific RNA editing

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.