Efficient in Vitro and in Vivo RNA Editing Via Recruitment of Endogenous ADARs Using Circular Guide RNAs

Overview

Journal Nat Biotechnol

Specialty Biotechnology

Date 2022 Feb 11

PMID 35145312

Authors

Dhruva Katrekar

James Yen

Yichen Xiang

Anushka Saha

Dario Meluzzi

Yiannis Savva

Prashant Mali

Affiliations

Soon will be listed here.

Abstract

Recruiting endogenous adenosine deaminases using exogenous guide RNAs to edit cellular RNAs is a promising therapeutic strategy, but editing efficiency and durability remain low using current guide RNA designs. In this study, we engineered circular ADAR-recruiting guide RNAs (cadRNAs) to enable more efficient programmable adenosine-to-inosine RNA editing without requiring co-delivery of any exogenous proteins. Using these cadRNAs, we observed robust and durable RNA editing across multiple sites and cell lines, in both untranslated and coding regions of RNAs, and high transcriptome-wide specificity. Additionally, we increased transcript-level specificity for the target adenosine by incorporating interspersed loops in the antisense domains, reducing bystander editing. In vivo delivery of cadRNAs via adeno-associated viruses enabled 53% RNA editing of the mPCSK9 transcript in C57BL/6J mice livers and 12% UAG-to-UGG RNA correction of the amber nonsense mutation in the IDUA-W392X mouse model of mucopolysaccharidosis type I-Hurler syndrome. cadRNAs enable efficient programmable RNA editing in vivo with diverse protein modulation and gene therapeutic applications.

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References

Melcher T, Maas S, Herb A, Sprengel R, Seeburg P, Higuchi M . A mammalian RNA editing enzyme. Nature. 1996; 379(6564):460-4. DOI: 10.1038/379460a0. View

Bass B, Weintraub H . An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell. 1988; 55(6):1089-98. DOI: 10.1016/0092-8674(88)90253-x. View

Bass B, Weintraub H . A developmentally regulated activity that unwinds RNA duplexes. Cell. 1987; 48(4):607-13. DOI: 10.1016/0092-8674(87)90239-x. View

Mannion N, Greenwood S, Young R, Cox S, Brindle J, Read D . The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 2014; 9(4):1482-94. PMC: 4542304. DOI: 10.1016/j.celrep.2014.10.041. View

Tomaselli S, Galeano F, Alon S, Raho S, Galardi S, Polito V . Modulation of microRNA editing, expression and processing by ADAR2 deaminase in glioblastoma. Genome Biol. 2015; 16:5. PMC: 4326501. DOI: 10.1186/s13059-014-0575-z. View

Schoft V, Schopoff S, Jantsch M . Regulation of glutamate receptor B pre-mRNA splicing by RNA editing. Nucleic Acids Res. 2007; 35(11):3723-32. PMC: 1920255. DOI: 10.1093/nar/gkm314. View

Wagner R, Smith J, Cooperman B, Nishikura K . A double-stranded RNA unwinding activity introduces structural alterations by means of adenosine to inosine conversions in mammalian cells and Xenopus eggs. Proc Natl Acad Sci U S A. 1989; 86(8):2647-51. PMC: 286974. DOI: 10.1073/pnas.86.8.2647. View

Nishikura K . A-to-I editing of coding and non-coding RNAs by ADARs. Nat Rev Mol Cell Biol. 2015; 17(2):83-96. PMC: 4824625. DOI: 10.1038/nrm.2015.4. View

Peng Z, Cheng Y, Chin-Ming Tan B, Kang L, Tian Z, Zhu Y . Comprehensive analysis of RNA-Seq data reveals extensive RNA editing in a human transcriptome. Nat Biotechnol. 2012; 30(3):253-60. DOI: 10.1038/nbt.2122. View

10.

Eggington J, Greene T, Bass B . Predicting sites of ADAR editing in double-stranded RNA. Nat Commun. 2011; 2:319. PMC: 3113232. DOI: 10.1038/ncomms1324. View

11.

Tan M, Li Q, Shanmugam R, Piskol R, Kohler J, Young A . Dynamic landscape and regulation of RNA editing in mammals. Nature. 2017; 550(7675):249-254. PMC: 5723435. DOI: 10.1038/nature24041. View

12.

Levanon E, Eisenberg E, Yelin R, Nemzer S, Hallegger M, Shemesh R . Systematic identification of abundant A-to-I editing sites in the human transcriptome. Nat Biotechnol. 2004; 22(8):1001-5. DOI: 10.1038/nbt996. View

13.

Montiel-Gonzalez M, Vallecillo-Viejo I, Yudowski G, Rosenthal J . Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing. Proc Natl Acad Sci U S A. 2013; 110(45):18285-90. PMC: 3831439. DOI: 10.1073/pnas.1306243110. View

14.

Wettengel J, Reautschnig P, Geisler S, Kahle P, Stafforst T . Harnessing human ADAR2 for RNA repair - Recoding a PINK1 mutation rescues mitophagy. Nucleic Acids Res. 2016; 45(5):2797-2808. PMC: 5389476. DOI: 10.1093/nar/gkw911. View

15.

Fukuda M, Umeno H, Nose K, Nishitarumizu A, Noguchi R, Nakagawa H . Construction of a guide-RNA for site-directed RNA mutagenesis utilising intracellular A-to-I RNA editing. Sci Rep. 2017; 7:41478. PMC: 5288656. DOI: 10.1038/srep41478. View

16.

Cox D, Gootenberg J, Abudayyeh O, Franklin B, Kellner M, Joung J . RNA editing with CRISPR-Cas13. Science. 2017; 358(6366):1019-1027. PMC: 5793859. DOI: 10.1126/science.aaq0180. View

17.

Merkle T, Merz S, Reautschnig P, Blaha A, Li Q, Vogel P . Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat Biotechnol. 2019; 37(2):133-138. DOI: 10.1038/s41587-019-0013-6. View

18.

Katrekar D, Chen G, Meluzzi D, Ganesh A, Worlikar A, Shih Y . In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat Methods. 2019; 16(3):239-242. PMC: 6395520. DOI: 10.1038/s41592-019-0323-0. View

19.

Qu L, Yi Z, Zhu S, Wang C, Cao Z, Zhou Z . Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat Biotechnol. 2019; 37(9):1059-1069. DOI: 10.1038/s41587-019-0178-z. View

20.

Monteleone L, Matthews M, Palumbo C, Thomas J, Zheng Y, Chiang Y . A Bump-Hole Approach for Directed RNA Editing. Cell Chem Biol. 2018; 26(2):269-277.e5. PMC: 6386613. DOI: 10.1016/j.chembiol.2018.10.025. View