Matsoukas, Ianis G.

Frontiers in Genetics

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Commentary: RNA editing with CRISPR-Cas13

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GENERAL COMMENTARYpublished: 18 April 2018doi: 10.3389/fgene.2018.00134Frontiers in Genetics | www.frontiersin.org 1 April 2018 | Volume 9 | Article 134Edited by:Graziano Pesole,Università degli Studi di Bari AldoMoro, ItalyReviewed by:Ernesto Picardi,Università degli Studi di Bari AldoMoro, Italy*Correspondence:Ianis G. Matsoukasi.matsoukas@bolton.ac.ukSpecialty section:This article was submitted toRNA,a section of the journalFrontiers in GeneticsReceived: 05 November 2017Accepted: 03 April 2018Published: 18 April 2018Citation:Matsoukas IG (2018) Commentary:RNA editing with CRISPR-Cas13.Front. Genet. 9:134.doi: 10.3389/fgene.2018.00134Commentary: RNA editing withCRISPR-Cas13Ianis G. Matsoukas*Faculty of Health and Wellbeing, School of Sport and Biomedical Sciences, University of Bolton, Bolton, United KingdomKeywords: ADAR-editing, RNA editing, CRISPR, targeted gene repair, Deaminase, TadAA commentary onRNA editing with CRISPR-Cas13by Cox, D. B. T., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J. et al.(2017). Science 358, 1019–1027. doi: 10.1126/science.aaq0180A paper recently published in Science (Cox et al., 2017) reports the possibility of editing RNAtranscripts to alter their coding potential in a programmable manner. It is proposed that the RNAEditing for Programmable A to I Replacement (REPAIR), the new genome-editing technology thattargets and alters RNA bases, offers a more temporary alternative to DNA editing.PROGRAMMABLE GENOME EDITING TOOLSGenome editing with programmable nucleases has become a powerful genetic tool. The term“programmable” refers to the ability to engineer the nuclease-based platforms for recognizingvarious target sites in different genomes. Many excellent reviews are available with regards togenome editing tools (Hsu et al., 2014; Kim and Kim, 2014; Cox et al., 2015; Kim, 2016). Therefore,the different genome editing-tools will not be discussed in great detail here.Genome editing tools include meganucleases (MN; Hafez and Hausner, 2012; Stoddard, 2014),zinc finger nucleases (ZFN; Carroll, 2011), transcription activator-like effector nucleases (TALENs;Boch et al., 2009; Boch, 2011), targetrons (Karberg et al., 2001), and the clustered regularlyinterspaced short palindromic repeat (CRISPR)-associated nuclease Cas9 (Bhaya et al., 2011;Jinek et al., 2012; Jiang et al., 2013). These genome editing tools can achieve precise genomemodifications by inducing targeted DNA double-strand breaks (DSBs). CRISPR system has rapidlygone from being a niche technology to a mainstream method (Figure 1A). Interestingly, ongoingimprovements of the CRISPR system have led to the development of powerful alternatives tostandard CRISPR technology (Abudayyeh et al., 2017; Gaudelli et al., 2017, Figure 1B). CRISPR(Barrangou et al., 2007), relies on the ability of CRISPR single guide RNAs (sgRNAs) to targetthe Cas9 endonuclease to precise genomic locations, where Cas9 introduces DSBs (Hsu et al., 2013;Doudna and Charpentier, 2014). MNs, ZFNs, and TALENs achieve sequence-specific DNA-bindingvia protein-DNA interactions (Kim and Kim, 2014), whereas CRISPR and targetrons are RNA-guided systems (Zimmerly et al., 1995; Jiang et al., 2013). One crucial concern when applying thesegenome editing tools is the potential of cleavage at non-targeted sites. This episode can be lethal orgenerate undesirable permanent alterations of the nucleotide sequences.Matsoukas Target and Edit RNAEXPLOITING A NOVEL CRISPR SYSTEMTO TARGET AND EDIT RNARNA editing is a posttranscriptional process through whichthe cellular machineries can make discrete changes to specificnucleoside sequences within a RNAmolecule (Gott and Emeson,2000; Bass, 2002). In humans, the most common type of RNAediting is the conversion of adenosine to inosine (A → I;Hogg et al., 2011; Wulff et al., 2011; Gilbert et al., 2016; Zhaoet al., 2017). This modification is mediated by two AdenosineDeaminases Acting on RNA (ADAR): ADAR1 and ADAR2. AsI is read as guanosine by the splicing and translation apparatuses,ADARs can also amend splicing patterns and modify amino-acidsequences (Patterson and Samuel, 1995; Gott and Emeson, 2000;Desterro et al., 2003; Hogg et al., 2011; Nishikura, 2016).Interestingly, a paper recently published in Science (Coxet al., 2017) reports the possibility of editing RNA transcriptsto alter their coding potential in a programmable manner.Cox et al. (2017) named the new system “RNA Editing forProgrammable A to I Replacement” (REPAIR; Figure 1C).This innovative article follows another important publicationFIGURE 1 | Programmable Genome Editing Tools. The conventional CRISPR DNA editor (A). CRISPR relies on the ability of CRISPR sgRNAs to target the Cas9endonuclease to precise genomic locations, where Cas9 introduces DSBs. Base editors borrow sgRNAs and Cas9 or other nucleases from CRISPR. However, baseeditors do not cut the double strand, but instead they chemically alter single bases with deaminase enzymes such as TadA (B, DNA base editor) and ADAR (C, RNAbase editor). Adapted from Doudna and Charpentier (2014), Cohen (2017), Cox et al. (2017), and Gaudelli et al. (2017).(Abudayyeh et al., 2017) from the same research groupshowing that a CRISPR system with an enzyme called Cas13acan target and cleave specific strands of RNA. Abudayyehet al. (2017) identified the Cas13a from Leptotrichia wadei(LwaCas13a). LwaCas13a was heterologously expressed inmammalian and plant cells for targeted knockdown of eitherreporter or endogenous transcripts with comparable levels ofknockdown as RNA interference, and improved specificity(Abudayyeh et al., 2017).To create REPAIR, Cox et al. (2017) systematically profiledthe CRISPR-Cas13 enzyme family for other potential “editor”candidates. They selected the Cas13b ortholog from Prevotella sp.P5-125 (PspCas13b), which was the most effective at inactivatingRNA. They successfully engineered a deactivated variant ofPspCas13b that still binds to specific nucleosides of RNA butlacks its “scissor-like” function. Then, the deactivated variantof PspCas13b was fused to ADAR2 deaminase domain, whichis involved (with ADAR1) in the A → I conversion in RNAtranscripts. Hence, in the novel system, the deactivated formof the Cas13b enzyme was able to recognize a target sequenceof RNA, whereas the ADAR2 element was performing the baseFrontiers in Genetics | www.frontiersin.org 2 April 2018 | Volume 9 | Article 134Matsoukas Target and Edit RNAconversion without cleaving the transcript or relying on thenative cellular apparatus.Cox et al. (2017) modified REPAIR to improve its specificityby creating the REPAIRv1 system. To demonstrate the broadapplicability of the REPAIRv1 system for RNA editing inmammalian cells, Cox et al. (2017) designed REPAIRv1 guidesagainst two disease relevant mutations: 878G>A (AVPR2W293X) in X-linked Nephrogenic diabetes insipidus, and1517G>A (FANCC W506X) in Fanconi anemia. In the cellline with the DNA containing the anemia mutation, REPAIRv1was able to correct 23% of the mutated RNA sequences. Inthe cell line containing the mutation causing the diabetes,REPAIRv1 was able to correct 35% of the mutated RNAsequences.Interestingly, Cox et al. (2017) further modified REPAIRv1to improve its specificity. The upgraded incarnation, REPAIRv2,consistently achieved the desired edit in 20–40%, and upto 51% of a targeted RNA without significant detection off-target activity. In addition, REPAIRv2 was able to reducethe detectable off-target edits from 18,385 to only 20 in thewhole transcriptome, providing dramatically higher specificitythan previously described RNA editing platforms (Stafforst andSchneider, 2012; Montiel-González et al., 2016).CONCLUDING REMARKSREPAIR presents a promising RNA editing platform with broadapplicability for biotechnology research and therapeutics. Coxet al. (2017) demonstrated the use of the PspCas13b enzyme asboth an RNA knockdown and RNA editing tool. Interestingly,the temporary nature of REPAIR-mediated edits will likely beuseful for treating diseases caused by temporary alterations. Forinstance, Cas13b could be fused to a variety of editing enzymesthat would allow a range of different sequence modifications.The REPAIR system is an excellent research tool. Introducingspecific sequence modifications into RNA molecules could allowto answer questions about alternative splicing mechanisms,translation, and even editing. In addition, by editing RNA ratherthan DNA, it might be possible to confer temporary, reversiblegenetic edits, rather than the CRISPR’s permanent genome edits.This would allow the potential for temporal control over editingoutcomes, as well as avoid the ethical issues that have arisenaround genome editing.However, due to RNA specific properties, the RNA baseeditors would have to be repeatedly administered to function as atherapeutic approach. In addition, despite the large number ofrecent findings and novelties to improve CRISPR via REPAIR(Cox et al., 2017), adenine base editors (Gaudelli et al., 2017),and other base editors (Komor et al., 2016), it could be severalyears before base-editing therapies enter clinical trials, and longeruntil it is clear whether the different base-editing strategies offeradvantages over existing gene therapies.AUTHOR CONTRIBUTIONSThe author confirms being the sole contributor of this work andapproved it for publication.FUNDINGResearch in the laboratory of IM is supported by a Jenkinson TIRIAward and the University of Bolton, UK.ACKNOWLEDGMENTSThe author would like to thank the editor for their support, andthe independent reviewers for their insightful comments on thearticle.REFERENCESAbudayyeh, O. O., Gootenberg, J. S., Essletzbichler, P., Han, S., Joung, J., Belanto,J. J., et al. (2017). RNA targeting with CRISPR-Cas13. 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Cell 82,545–554. doi: 10.1016/0092-8674(95)90027-6Conflict of Interest Statement: The author declares that the research wasconducted in the absence of any commercial or financial relationships that couldbe construed as a potential conflict of interest.The reviewer EP and handling Editor declared their shared affiliation.Copyright © 2018 Matsoukas. This is an open-access article distributed under theterms of the Creative Commons Attribution License (CC BY). The use, distributionor reproduction in other forums is permitted, provided the original author(s) and thecopyright owner are credited and that the original publication in this journal is cited,in accordance with accepted academic practice. No use, distribution or reproductionis permitted which does not comply with these terms.Frontiers in Genetics | www.frontiersin.org 4 April 2018 | Volume 9 | Article 134

Ianis G. Matsoukas

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Commentary: RNA editing with CRISPR-Cas13

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