期刊
METHODS
卷 205, 期 -, 页码 158-166出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ymeth.2022.06.011
关键词
AdenosinedeaminasesactingonRNA(ADARs); Adenosinetoinosine(A-to-I)RNAediting; GuideRNAs; CircularguideRNAs; Split-proteins; Transcriptomeengineering
资金
- UCSD Institutional Funds, NIH [R01HG009285, R01CA222826, R01GM123313]
- Department of Defense Grant [W81XWH-22-1-0401]
- Norn Group
In this chapter, two strategies for achieving site-specific A-to-I RNA editing are presented: recruitment of endogenous ADARs and recruitment of exogenous ADARs. The use of circular adRNAs and a split-ADAR2 system enable robust and specific editing in various RNA editing applications.
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 & PRIME; guanosine. We anticipate the described methods should facilitate RNA editing applications across research and biotechnology settings.
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