Viewpoint Cite This: Biochemistry XXXX, XXX, XXX−XXX
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Expanding the Chemical Scope of RNA Base Editors Simone Rauch†,‡ and Bryan C. Dickinson*,† †
Department of Chemistry, University of Chicago, Chicago, Illinois 60637, United States Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois 60637, United States
‡
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NA has emerged as an exciting therapeutic target for disease treatment. Genetic therapies targeting RNA retain the same programmable and selective attributes as those targeting DNA. However, unlike DNA-targeting strategies, which result in permanent changes to the genetic code, RNAtargeting therapies can in principle be dosed, providing an element of temporal control that more closely emulates traditional regimens for biological therapeutics. This additional control should provide both enhanced safety windows and the ability to target a larger number of diseases as treatments can be halted or adjusted in response to any emergent side effects. Additionally, because gene expression is regulated by a diverse array of mechanisms at the RNA level, various modes of intervention and control can be employed. In particular, RNA editing represents a unique opportunity to correct diseasecausing nucleotide mutations, and resultant protein sequence errors, at the transcript level. In the past, most of the work in this area has been focused on adenosine-to-inosine (A-to-I) editing mediated by site-specific recruitment of human ADAR. Abudayyeh et al.1 have now expanded the scope of RNA editing by engineering a programmable cytosine-to-uridine (Cto-U) RNA base editor, increasing the number of diseasecausing mutations that can be corrected at the RNA level (Figure 1A). RNA editing is a common post-transcriptional modification in mammalian cells and serves to alter the genetic code at the RNA level. Chemical deamination, most notably deamination of adenosine to inosine, or cytosine to uracil, is the most common mode of editing. A variety of programmable RNA base editors that mediate site-specific deamination reactions have been developed and deployed in in vivo models.2 Thus far, a majority of these programmable editors have focused on A-to-I editing, taking advantage of either an evolved, hyperactive variant of the catalytic domain of the human Ato-I editing enzyme hADAR, which has low sequence and structural requirements, or the recruitment of endogenous ADARs. Hairpin binding protein interactions fused to ADARs, SNAP tag-fused ADARs, and dCas13 have all been used to deliver ADARs to target sites and induce edits (Figure 1B). To overcome off-target effects caused by overexpressing hADAR, a variety of technologies have been developed to deliver endogenous ADAR to a target RNA, precluding the need for ADAR overexpression. For example, chemically modified3 recruiting oligonucleotides can redirect endogenous ADAR to a target site on an RNA of interest. While chemical modifications increase the stability and therefore the performance of these approaches, the modifications can also cause immunogenic responses when administered repeatedly, potentially limiting their therapeutic applications. Consequently, Qu et al. developed engineered ADAR-recruiting RNAs (arRNAs),4 which are not chemically modified and can therefore be © XXXX American Chemical Society
Figure 1. Overview of current approaches for RNA editing. (A) Most RNA editing technologies utilized to date deploy ADAR-mediated Ato-I editors. Other edits, such as C to U, also occur in natural systems, for example, by the APOBEC proteins. However, these proteins tend to have specific structural requirements of the target RNA. Now, an evolved ADAR variant allows for C-to-U edits on target RNAs, expanding the chemical scope of possible RNA edits. (B) A variety of methods have been developed to deliver RNA editors to target transcripts.
genetically encoded. Expression of arRNAs resulted in efficient A-to-I editing in human primary cell lines and several diseaserelevant contexts, including restoration of transcriptional regulatory activity of mutant TP53W53X and recovery of IDUA activity in Hurler syndrome, emphasizing the therapeutic potential of this approach. Received: August 5, 2019
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DOI: 10.1021/acs.biochem.9b00676 Biochemistry XXXX, XXX, XXX−XXX
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Biochemistry ORCID
Guided A-to-I editors can correct only G-to-A mutations, which account for one-quarter of all potential nucleotide point mutations. In their newly published work, Abudayyeh et al.1 expand the RNA editing scope via development of the first programmable cytosine-to-uracil (C-to-U) RNA editor, RNA Editing for Specific C-to-U Exchange (RESCUE). RESCUE is guided by a catalytically inactive “dead” Cas13 protein fused to an evolved variant of hyperactive hADAR2 (ADAR2dd). Although natural cytosine deamination enzymes have been harnessed for programmable DNA editing, these enzymes act on single-stranded substrates and with appreciable off-target activity. To address these hurdles, the authors evolved the commonly used hyperactive hADAR2 variant, previously employed in the REPAIR platform, to deaminate cytosine to uracil. On the basis of structural similarities with the Escherichia coli cytidine deaminase, the authors first executed three rounds of rational mutagenesis of ADAR2dd, followed by 16 rounds of random mutagenesis along the entire protein, yielding RESCUEr16, a cytosine deaminase with increased activity across most sequence contexts. After extensive biochemical characterization, RESCUE was tested on endogenous transcripts in HEK293FT cells using bulk sequencing of nine genes and 24 synthetic diseaserelevant mutation targets. RESCUE yielded editing rates of ≤42% on these endogenous transcripts and achieved higher editing rates when the mismatch position was immediately preceded with a U or A. Next, the authors deployed RESCUE to activate the STAT and Wnt/β-catenin pathway by editing a key phosphorylated residue. Upon successful cytosine editing, they observed a ≤5-fold induction of Wnt/β-catenin signaling and an increased level of cell growth in both model HEK293FT and human umbilical vein endothelial cells. Because the A-to-I editing activity was preserved through the evolution, the system is capable of multiplexed editing but can also produce off-target edits. By performing another round of rational mutagenesis at residues interacting with the RNA target, the authors produced a variant, RESCUE-S, with retained editing performance but higher specificity. RNA editing by delivering exogenous enzymes that potentially lead to a large number of off-target edits throughout the entire transcriptome continues to be a major challenge with protein-based editing systems. To combat this limitation, a variety of approaches to the delivery endogenous ADAR have been developed. However, these strategies are limited by the availability of endogenous editing enzymes and in some cases still require supplemental hADAR expression via the interferon pathway. Additionally, heavily modified RNA oligonucleotides can elicit immunogenic responses when repeatedly administered. Continuing advances expanding the functionality of RNA editing protein systems can be integrated with a variety of delivery systems. For example, we recently developed CIRTS,5 a human protein-based delivery system that can deliver a range of effector proteins, including ADARs, by gRNA-directed protein delivery, potentially alleviating immunogenicity concerns. Ongoing expansion of the scope of RNA editing and RNA effector proteins by directed evolution will continue to open up new methods for controlling and modulating gene expression at the RNA level and exciting new opportunities for therapeutic intervention.
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Simone Rauch: 0000-0003-1511-269X Bryan C. Dickinson: 0000-0002-9616-1911 Funding
This work was supported by the National Institute of General Medical Sciences of the National Institutes of Health (R35 GM119840). Notes
The authors declare the following competing financial interest(s): The authors have a provisional patent on the CIRTS technology, which includes base editing.
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REFERENCES
(1) Abudayyeh, O. O., Gootenberg, J. S., Franklin, B., Koob, J., Kellner, M. J., Ladha, A., Joung, J., Kirchgatterer, P., Cox, D. B. T., and Zhang, F. (2019) A cytosine deaminase for programmable single-base RNA editing. Science 365, 382−386. (2) Katrekar, D., Chen, G., Meluzzi, D., Ganesh, A., Worlikar, A., Shih, Y. R., Varghese, S., and Mali, P. (2019) In vivo RNA editing of point mutations via RNA-guided adenosine deaminases. Nat. Methods 16, 239−242. (3) Merkle, T., Merz, S., Reautschnig, P., Blaha, A., Li, Q., Vogel, P., Wettengel, J., Li, J. B., and Stafforst, T. (2019) Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides. Nat. Biotechnol. 37, 133−138. (4) Qu, L., Yi, Z., Zhu, S., Wang, C., Cao, Z., Zhou, Z., Yuan, P., Yu, Y., Tian, F., Liu, Z., Bao, Y., Zhao, Y., and Wei, W. (2019) Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs. Nat. Biotechnol., DOI: 10.1038/s41587-019-0178z. (5) Rauch, S., He, E., Srienc, M., Zhou, H., Zhang, Z., and Dickinson, B. C. (2019) Programmable RNA-Guided RNA Effector Proteins Built from Human Parts. Cell 178, 122−134.e12.
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DOI: 10.1021/acs.biochem.9b00676 Biochemistry XXXX, XXX, XXX−XXX
