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• W4311468629 abstract "•RESTART relies on guide snoRNA to revert PTC-induced translation termination•The minor DKC1-isoform3 greatly increases the RNA-editing efficiency of RESTART•RESTART enables efficient pseudouridylation in cell lines and primary cells•RESTART mediates functional PTC readthrough in disease-relevant contexts Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics. Nonsense mutations, accounting for >20% of disease-associated mutations, lead to premature translation termination. Replacing uridine with pseudouridine in stop codons suppresses translation termination, which could be harnessed to mediate readthrough of premature termination codons (PTCs). Here, we present RESTART, a programmable RNA base editor, to revert PTC-induced translation termination in mammalian cells. RESTART utilizes an engineered guide snoRNA (gsnoRNA) and the endogenous H/ACA box snoRNP machinery to achieve precise pseudouridylation. We also identified and optimized gsnoRNA scaffolds to increase the editing efficiency. Unexpectedly, we found that a minor isoform of pseudouridine synthase DKC1, lacking a C-terminal nuclear localization signal, greatly improved the PTC-readthrough efficiency. Although RESTART induced restricted off-target pseudouridylation, they did not change the coding information nor the expression level of off-targets. Finally, RESTART enables robust pseudouridylation in primary cells and achieves functional PTC readthrough in disease-relevant contexts. Collectively, RESTART is a promising RNA-editing tool for research and therapeutics. Nonsense mutations are generated from single base-pair substitutions and introduce in-frame premature termination codons (PTCs) in mRNA coding regions, resulting in the production of truncated and often nonfunctional proteins, degradation of PTC-containing mRNA by nonsense-mediated mRNA decay (NMD), and eventually genetic disorders.1Mendell J.T. Dietz H.C. When the message goes awry: disease-producing mutations that influence mRNA content and performance.Cell. 2001; 107: 411-414https://doi.org/10.1016/s0092-8674(01)00583-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar,2Linde L. Kerem B. Introducing sense into nonsense in treatments of human genetic diseases.Trends Genet. 2008; 24: 552-563https://doi.org/10.1016/j.tig.2008.08.010Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar,3Cartegni L. Chew S.L. Krainer A.R. Listening to silence and understanding nonsense: exonic mutations that affect splicing.Nat. Rev. Genet. 2002; 3: 285-298https://doi.org/10.1038/nrg775Crossref PubMed Scopus (1769) Google Scholar According to the Human Gene Mutation Database (HGMD; www.hgmd.org), more than 20% of the disease-associated single base-pair substitutions are nonsense mutations, which account for approximately 11% of all described gene lesions causing human inherited diseases.4Mort M. Ivanov D. Cooper D.N. Chuzhanova N.A. A meta-analysis of nonsense mutations causing human genetic disease.Hum. Mutat. 2008; 29: 1037-1047https://doi.org/10.1002/humu.20763Crossref PubMed Scopus (275) Google Scholar,5Stenson P.D. Mort M. Ball E.V. Howells K. Phillips A.D. Thomas N.S. Cooper D.N. The Human Gene Mutation Database: 2008 update.Genome Med. 2009; 1: 13Crossref PubMed Scopus (701) Google Scholar For inherited diseases in individual cases, the incidence of nonsense mutations can also range from 5% to 70%.1Mendell J.T. Dietz H.C. When the message goes awry: disease-producing mutations that influence mRNA content and performance.Cell. 2001; 107: 411-414https://doi.org/10.1016/s0092-8674(01)00583-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar Therefore, every effort should be made to develop therapeutic strategies to tackle PTC-mediated diseases. Several RNA-targeting approaches exist to treat genetic disorders caused by nonsense mutations. Splice-switching antisense oligonucleotides can be used to splice out PTC-containing exons in frame but may encode a nonfunctional or partially functional protein.6Havens M.A. Hastings M.L. Splice-switching antisense oligonucleotides as therapeutic drugs.Nucleic Acids Res. 2016; 44: 6549-6563https://doi.org/10.1093/nar/gkw533Crossref PubMed Scopus (276) Google Scholar,7Barny I. Perrault I. Michel C. Goudin N. Defoort-Dhellemmes S. Ghazi I. Kaplan J. Rozet J.M. Gerard X. AON-mediated exon skipping to bypass protein truncation in retinal dystrophies due to the recurrent CEP290 c.4723A > T mutation. Fact or Fiction?.Genes. 2019; 10: 368https://doi.org/10.3390/genes10050368Crossref PubMed Scopus (12) Google Scholar Small molecules (e.g., aminoglycoside antibiotics and oxadiazole derivatives) have been identified to induce PTC readthrough via oral administration8Howard M. Frizzell R.A. Bedwell D.M. Aminoglycoside antibiotics restore CFTR function by overcoming premature stop mutations.Nat. Med. 1996; 2: 467-469https://doi.org/10.1038/nm0496-467Crossref PubMed Scopus (415) Google Scholar,9Welch E.M. Barton E.R. Zhuo J. Tomizawa Y. Friesen W.J. Trifillis P. Paushkin S. Patel M. Trotta C.R. Hwang S. et al.PTC124 targets genetic disorders caused by nonsense mutations.Nature. 2007; 447: 87-91https://doi.org/10.1038/nature05756Crossref PubMed Scopus (887) Google Scholar; however, they lack specificity and show toxicity after prolonged use.10Dabrowski M. Bukowy-Bieryllo Z. Zietkiewicz E. Advances in therapeutic use of a drug-stimulated translational readthrough of premature termination codons.Mol. Med. Camb. Mass. 2018; 24: 25https://doi.org/10.1186/s10020-018-0024-7Crossref PubMed Scopus (81) Google Scholar Engineered tRNAs have been used to read through PTCs, but they also lack sequence specificity.11Shi N. Yang Q. Zhang H. Lu J. Lin H. Yang X. Abulimiti A. Cheng J. Wang Y. Tong L. et al.Restoration of dystrophin expression in mice by suppressing a nonsense mutation through the incorporation of unnatural amino acids.Nat. Biomed. Eng. 2022; 6: 195-206https://doi.org/10.1038/s41551-021-00774-1Crossref PubMed Scopus (14) Google Scholar,12Lueck J.D. Yoon J.S. Perales-Puchalt A. Mackey A.L. Infield D.T. Behlke M.A. Pope M.R. Weiner D.B. Skach W.R. McCray Jr., P.B. Ahern C.A. Engineered transfer RNAs for suppression of premature termination codons.Nat. Commun. 2019; 10: 822https://doi.org/10.1038/s41467-019-08329-4Crossref PubMed Scopus (61) Google Scholar Leveraging the deamination activity of ADAR and APOBEC proteins, several A-to-I (inosine) and/or C-to-U RNA base editors have been developed.13Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027https://doi.org/10.1126/science.aaq0180Crossref PubMed Scopus (991) Google Scholar,14Merkle T. Merz S. Reautschnig P. Blaha A. Li Q. Vogel P. Wettengel J. Li J.B. Stafforst T. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.Nat. Biotechnol. 2019; 37: 133-138https://doi.org/10.1038/s41587-019-0013-6Crossref PubMed Scopus (140) Google Scholar,15Qu L. Yi Z. Zhu S. Wang C. Cao Z. Zhou Z. Yuan P. Yu Y. Tian F. Liu Z. et al.Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.Nat. Biotechnol. 2019; 37: 1059-1069https://doi.org/10.1038/s41587-019-0178-zCrossref PubMed Scopus (120) Google Scholar,16Katrekar D. Chen G. Meluzzi D. Ganesh A. Worlikar A. Shih Y.R. Varghese S. Mali P. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.Nat. Methods. 2019; 16: 239-242https://doi.org/10.1038/s41592-019-0323-0Crossref PubMed Scopus (103) Google Scholar,17Vogel P. Moschref M. Li Q. Merkle T. Selvasaravanan K.D. Li J.B. Stafforst T. Efficient and precise editing of endogenous transcripts with SNAP-tagged ADARs.Nat. Methods. 2018; 15: 535-538https://doi.org/10.1038/s41592-018-0017-zCrossref PubMed Scopus (88) Google Scholar,18Huang X. Lv J. Li Y. Mao S. Li Z. Jing Z. Sun Y. Zhang X. Shen S. Wang X. et al.Programmable C-to-U RNA editing using the human APOBEC3A deaminase.EMBO J. 2020; 39: e104741https://doi.org/10.15252/embj.2020104741Crossref PubMed Scopus (16) Google Scholar,19Abudayyeh O.O. Gootenberg J.S. Franklin B. Koob J. Kellner M.J. Ladha A. Joung J. Kirchgatterer P. Cox D.B.T. Zhang F. A cytosine deaminase for programmable single-base RNA editing.Science. 2019; 365: 382-386https://doi.org/10.1126/science.aax7063Crossref PubMed Scopus (228) Google Scholar,20Stafforst T. Schneider M.F. An RNA-deaminase conjugate selectively repairs point mutations.Angew. Chem. Int. Ed. Engl. 2012; 51: 11166-11169https://doi.org/10.1002/anie.201206489Crossref PubMed Scopus (89) Google Scholar,21Montiel-Gonzalez M.F. Vallecillo-Viejo I. Yudowski G.A. Rosenthal J.J. Correction of mutations within the cystic fibrosis transmembrane conductance regulator by site-directed RNA editing.Proc. Natl. Acad. Sci. USA. 2013; 110: 18285-18290https://doi.org/10.1073/pnas.1306243110Crossref PubMed Scopus (138) Google Scholar,22Rauch S. He E. Srienc M. Zhou H. Zhang Z. Dickinson B.C. Programmable RNA-guided RNA effector proteins built from human parts.Cell. 2019; 178 (122.e12–134.e12)https://doi.org/10.1016/j.cell.2019.05.049Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar,23Reautschnig P. Wahn N. Wettengel J. Schulz A.E. Latifi N. Vogel P. Kang T.W. Pfeiffer L.S. Zarges C. Naumann U. et al.CLUSTER guide RNAs enable precise and efficient RNA editing with endogenous ADAR enzymes in vivo.Nat. Biotechnol. 2022; 40: 759-768https://doi.org/10.1038/s41587-021-01105-0Crossref PubMed Scopus (23) Google Scholar,24Yi Z. Qu L. Tang H. Liu Z. Liu Y. Tian F. Wang C. Zhang X. Feng Z. Yu Y. et al.Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo.Nat. Biotechnol. 2022; 40: 946-955https://doi.org/10.1038/s41587-021-01180-3Crossref PubMed Scopus (32) Google Scholar,25Katrekar D. Yen J. Xiang Y. Saha A. Meluzzi D. Savva Y. Mali P. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs.Nat. Biotechnol. 2022; 40: 938-945https://doi.org/10.1038/s41587-021-01171-4Crossref PubMed Scopus (36) Google Scholar,26Xu C. Zhou Y. Xiao Q. He B. Geng G. Wang Z. Cao B. Dong X. Bai W. Wang Y. et al.Programmable RNA editing with compact CRISPR-Cas13 systems from uncultivated microbes.Nat. Methods. 2021; 18: 499-506https://doi.org/10.1038/s41592-021-01124-4Crossref PubMed Scopus (93) Google Scholar,27Marina R.J. Brannan K.W. Dong K.D. Yee B.A. Yeo G.W. Evaluation of engineered CRISPR-Cas-mediated systems for site-specific RNA editing.Cell Rep. 2020; 33: 108350https://doi.org/10.1016/j.celrep.2020.108350Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,28Porto E.M. Komor A.C. Slaymaker I.M. Yeo G.W. Base editing: advances and therapeutic opportunities.Nat. Rev. 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RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027https://doi.org/10.1126/science.aaq0180Crossref PubMed Scopus (991) Google Scholar; directed evolution of ADAR2 also endows it with the ability to possess simultaneous C-to-U editing activity in RESCUE.19Abudayyeh O.O. Gootenberg J.S. Franklin B. Koob J. Kellner M.J. Ladha A. Joung J. Kirchgatterer P. Cox D.B.T. Zhang F. A cytosine deaminase for programmable single-base RNA editing.Science. 2019; 365: 382-386https://doi.org/10.1126/science.aax7063Crossref PubMed Scopus (228) Google Scholar RESTORE and LEAPER utilize a short gRNA to recruit the endogenous ADAR protein for RNA editing.14Merkle T. Merz S. Reautschnig P. Blaha A. Li Q. Vogel P. Wettengel J. Li J.B. Stafforst T. Precise RNA editing by recruiting endogenous ADARs with antisense oligonucleotides.Nat. Biotechnol. 2019; 37: 133-138https://doi.org/10.1038/s41587-019-0013-6Crossref PubMed Scopus (140) Google Scholar,15Qu L. Yi Z. Zhu S. Wang C. Cao Z. Zhou Z. Yuan P. Yu Y. Tian F. Liu Z. et al.Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.Nat. Biotechnol. 2019; 37: 1059-1069https://doi.org/10.1038/s41587-019-0178-zCrossref PubMed Scopus (120) Google Scholar Sequence-specific A-to-I editing has also been achieved in mouse models of human disease.16Katrekar D. Chen G. Meluzzi D. Ganesh A. Worlikar A. Shih Y.R. Varghese S. Mali P. In vivo RNA editing of point mutations via RNA-guided adenosine deaminases.Nat. Methods. 2019; 16: 239-242https://doi.org/10.1038/s41592-019-0323-0Crossref PubMed Scopus (103) Google Scholar,24Yi Z. Qu L. Tang H. Liu Z. Liu Y. Tian F. Wang C. Zhang X. Feng Z. Yu Y. et al.Engineered circular ADAR-recruiting RNAs increase the efficiency and fidelity of RNA editing in vitro and in vivo.Nat. Biotechnol. 2022; 40: 946-955https://doi.org/10.1038/s41587-021-01180-3Crossref PubMed Scopus (32) Google Scholar,25Katrekar D. Yen J. Xiang Y. Saha A. Meluzzi D. Savva Y. Mali P. Efficient in vitro and in vivo RNA editing via recruitment of endogenous ADARs using circular guide RNAs.Nat. Biotechnol. 2022; 40: 938-945https://doi.org/10.1038/s41587-021-01171-4Crossref PubMed Scopus (36) Google Scholar Nevertheless, these RNA base editors have potential proximal and distal off-target editing problems.27Marina R.J. Brannan K.W. Dong K.D. Yee B.A. Yeo G.W. Evaluation of engineered CRISPR-Cas-mediated systems for site-specific RNA editing.Cell Rep. 2020; 33: 108350https://doi.org/10.1016/j.celrep.2020.108350Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar,31Vogel P. Stafforst T. Critical review on engineering deaminases for site-directed RNA editing.Curr. Opin. Biotechnol. 2019; 55: 74-80https://doi.org/10.1016/j.copbio.2018.08.006Crossref PubMed Scopus (35) Google Scholar Therefore, specific and safe strategies that can efficiently edit RNA bases with minimal consequences by off-target editing are highly desired. As the most abundant modification in RNA, pseudouridine (Ψ) possesses a similar base-pairing property as uridine (U)32Ge J. Yu Y.T. RNA pseudouridylation: new insights into an old modification.Trends Biochem. Sci. 2013; 38: 210-218https://doi.org/10.1016/j.tibs.2013.01.002Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar,33Roundtree I.A. Evans M.E. Pan T. He C. Dynamic RNA modifications in gene expression regulation.Cell. 2017; 169: 1187-1200https://doi.org/10.1016/j.cell.2017.05.045Abstract Full Text Full Text PDF PubMed Scopus (1641) Google Scholar,34Li X. Ma S. Yi C. Pseudouridine: the fifth RNA nucleotide with renewed interests.Curr. Opin. Chem. Biol. 2016; 33: 108-116https://doi.org/10.1016/j.cbpa.2016.06.014Crossref PubMed Scopus (96) Google Scholar and is catalyzed via stand-alone synthases or an RNA-guided enzyme complex.35Karijolich J. Yi C. Yu Y.T. Transcriptome-wide dynamics of RNA pseudouridylation.Nat. Rev. Mol. Cell Biol. 2015; 16: 581-585https://doi.org/10.1038/nrm4040Crossref PubMed Scopus (83) Google Scholar For instance, site-specific pseudouridylation of rRNA and snRNA is catalyzed primarily by H/ACA box snoRNP (ribonucleoprotein).36Kiss T. Fayet-Lebaron E. Jády B.E. Box H/ACA small ribonucleoproteins.Mol. Cell. 2010; 37: 597-606https://doi.org/10.1016/j.molcel.2010.01.032Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar In the complex, H/ACA snoRNA functions as the RNA guide through base-pairing with target RNA and associates with four evolutionarily conserved core proteins DKC1, NHP2, NOP10, and GAR1. Among them, DKC1 is the Ψ synthase that provides catalytic activity. Although Ψ only causes negligible alterations to the coding properties of sense codons,37Eyler D.E. Franco M.K. Batool Z. Wu M.Z. Dubuke M.L. Dobosz-Bartoszek M. Jones J.D. Polikanov Y.S. Roy B. Koutmou K.S. Pseudouridinylation of mRNA coding sequences alters translation.Proc. Natl. Acad. Sci. USA. 2019; 116: 23068-23074Crossref PubMed Scopus (82) Google Scholar Yu and colleagues reported that replacing U with Ψ in all three stop codons can unexpectedly suppress translation termination.38Karijolich J. Yu Y.T. Converting nonsense codons into sense codons by targeted pseudouridylation.Nature. 2011; 474: 395-398https://doi.org/10.1038/nature10165Crossref PubMed Scopus (238) Google Scholar,39Fernández I.S. Ng C.L. Kelley A.C. Wu G. Yu Y.T. Ramakrishnan V. Unusual base pairing during the decoding of a stop codon by the ribosome.Nature. 2013; 500: 107-110https://doi.org/10.1038/nature12302Crossref PubMed Scopus (122) Google Scholar In yeast, artificially designed snoRNA has been used to direct pseudouridylation at the PTC sites, with an ∼5% modification efficiency38Karijolich J. Yu Y.T. Converting nonsense codons into sense codons by targeted pseudouridylation.Nature. 2011; 474: 395-398https://doi.org/10.1038/nature10165Crossref PubMed Scopus (238) Google Scholar,40Huang C. Wu G. Yu Y.T. Inducing nonsense suppression by targeted pseudouridylation.Nat. Protoc. 2012; 7: 789-800https://doi.org/10.1038/nprot.2012.029Crossref PubMed Scopus (18) Google Scholar; however, in contrast to extensive studies in yeast and archaea, our understanding of the targeting and activity of H/ACA box snoRNP in mammals is limited. In addition, unlike stop codon readthrough promoted by RNA sequence or structure, Ψ-mediated stop codon readthrough is independent of the sequence context.41Adachi H. Yu Y.T. Pseudouridine-mediated stop codon readthrough in S. cerevisiae is sequence context-independent.RNA. 2020; 26: 1247-1256https://doi.org/10.1261/rna.076042.120Crossref PubMed Google Scholar Thus, snoRNA-guided pseudouridylation at PTCs offers a promising opportunity for RNA-based therapy against nonsense mutation-induced human diseases. In this study, we developed RNA editing to specific transcripts for pseudouridine-mediated PTC readthrough (RESTART) to achieve efficient and precise Ψ modification in mammalian cells. RESTART leverages the H/ACA box snoRNP machinery for RNA-targeting and editing and hence is CRISPR-free. We report two systems, RESTARTv1 and RESTARTv2; the former involves only one designed guide snoRNA (gsnoRNA) and the latter contains an additional naturally occurring but minor isoform of DKC1. Both systems demonstrate broad applicability for readthrough of disease-relevant nonsense mutations, without causing undesired consequences owing to off-target editing. H/ACA snoRNAs contain two hairpins followed respectively by the H and ACA box motifs (Figure 1A), and we designed both hairpins of gsnoRNAs to target a given PTC site in mRNA. To assess the efficiency of PTC readthrough, we generated a Venus reporter (Reporter-1), in which an amber codon (TAG) was inserted to prematurely terminate Venus translation (Figure 1B). One would expect to see fluorescence if gsnoRNA-mediated pseudouridylation occurs at the amber codon and leads to readthrough. PTC-readthrough events were measured by a high-content imaging system and quantified by comparison with the positive control group (Figures 1C and 1D; STAR Methods). We selected 4 snoRNAs with high expression levels in human cells42Jorjani H. Kehr S. Jedlinski D.J. Gumienny R. Hertel J. Stadler P.F. Zavolan M. Gruber A.R. An updated human snoRNAome.Nucleic Acids Res. 2016; 44: 5068-5082https://doi.org/10.1093/nar/gkw386Crossref PubMed Scopus (162) Google Scholar and detected 5.2% and 5.0% Venus positive cells (compared with the positive control) from cells transfected with host-gACA19 and host-gE2, respectively, indicating gsnoRNA-mediated PTC readthrough (Figure S1A). The other two gsnoRNAs displayed negligible signals. An RNA secondary structure prediction revealed that the gsnoRNA scaffolds of gACA19 and gE2 appear to be more stable than gACA44 and gACA27 (Table S1), possibly related to their ability to guide pseudouridylation. Since in the human genome, more than 90% snoRNA genes are encoded in introns,43Dieci G. Preti M. Montanini B. Eukaryotic snoRNAs: a paradigm for gene expression flexibility.Genomics. 2009; 94: 83-88https://doi.org/10.1016/j.ygeno.2009.05.002Crossref PubMed Scopus (223) Google Scholar we compared the gsnoRNAs located within the introns of their respective host genes or HBB gene44Richard P. Kiss A.M. Darzacq X. Kiss T. Cotranscriptional recognition of human intronic box H/ACA snoRNAs occurs in a splicing-independent manner.Mol. Cell. Biol. 2006; 26: 2540-2549https://doi.org/10.1128/MCB.26.7.2540-2549.2006Crossref PubMed Scopus (59) Google Scholar and hU6/hU1 promoter-driven gsnoRNAs. We found gsnoRNAs driven by the small RNA promoter displayed higher efficiency in mediating PTC readthrough compared with intron-embedded gsnoRNAs, thus we selected gsnoRNAs driven by the hU6 promoter as a universal vector for expression (Figures 1C, 1D, and S1A–S1C). We also showed that both box H and box ACA have an important role in gsnoRNA function, since mutating or deleting the box H/ACA greatly reduced or even abolished readthrough activity (Figure S1D). To determine whether endogenous DKC1 protein is responsible for the gsnoRNA-mediated PTC readthrough, we carried out RESTART in DKC1 stable knockdown (DKC1-KD) cells (Figure 1E). No PTC readthrough was observed for gsnoRNAs in DKC1-KD cells, whereas the gsnoRNAs clearly activated the expression of Venus in the control groups (Figure 1F), demonstrating a key role of endogenous DKC1 in mediating stop codon readthrough (Figure 1A). Collectively, these observations showed that the designed gsnoRNAs are capable of inducing PTC readthrough of targeted RNA transcripts, which is dependent on the endogenous pseudouridylation complex. To identify optimal gsnoRNA scaffolds, we selected four additional snoRNAs (gACA3, gACA17, gACA2b, and gACA36) with the stable secondary structures predicted by RNAfold for further characterization, in addition to gACA19 (Figure 2A; Table S1). Among them, gACA36 and gACA2b outcompeted gACA19 and displayed the highest efficiencies of PTC readthrough (relative Venus positive cells: 13.7% and 12.2%, respectively) (Figures 2B and 2C). The differential readthrough efficiency may be caused by the expression level as well as additional factors of gsnoRNA scaffolds (Figure S1E). To investigate the roles of two individual hairpins in gsnoRNAs, we introduced mutations in either the guide elements of the 5′ or 3′ hairpin (Figure 2A; Table S1). We found although two hairpins of gsnoRNA target the same site, the editing efficiency could be largely dominated by one of the two hairpins (Figures 2D, 2E, and S1F). We further attempted to improve the PTC readthrough efficiency by engineering gsnoRNA scaffolds (Figures 2D, 2E, and S1G; Table S1). Given that RNA polymerase III terminates transcription at short polyUs stretch, we introduced single base mutation to the “UUUU” sequence in the apical loop of gACA19 (Figure 2A; Table S1). As expected, the improved expression of the engineered gACA19 resulted in improvement of PTC-readthrough efficiency (Figures 2D, 2E, S1E, and S1H). It has also been reported that the distance between the target U and the H/ACA box is about 14–15 nt for most natural snoRNAs, which is critical for the guide function.36Kiss T. Fayet-Lebaron E. Jády B.E. Box H/ACA small ribonucleoproteins.Mol. Cell. 2010; 37: 597-606https://doi.org/10.1016/j.molcel.2010.01.032Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar The distance in the gACA19 3′ hairpin is 13 nt; hence, we inserted a single base after U115 of gACA19 (Figure 2A; Table S1) and found that gACA19-3addG increased the efficiencies to 1.4-fold compared with unmodified gACA19 (Figures 2D and 2E). To make the guide elements more accessible, we inserted two nucleotides after U8 in the 5′ hairpin of gACA19 (Figure 2A; Table S1) and gACA19-5addCU increased the PTC-readthrough efficiencies to 1.6-fold (Figures 2D and 2E). We also combined the above mutations of gACA19, engineered the gACA36 scaffolds and tandemly expressed the two gsnoRNAs, but the efficiency of PTC readthrough was not further improved (Figures S1F, S1I, and S1J; Table S1). We further confirmed RESTART-mediated readthrough using two additional reporters Reporter-2 and Reporter-3 (Figures S1K and S1L). gsnoRNAs showed increased PTC-readthrough efficiencies in Reporter-3 (relative EGFP positive cells: ∼30%, ∼2-fold compared with Reporter-1 and Reporter-2) (Figures 2B, 2D, S1K, and S1L), possibly because gsnoRNA is arranged in tandem with the reporter sequence (Figure S1L). We also ruled out the possibility that the expression of the reporter transcripts was perturbed by gsnoRNAs (Figure S1M). Altogether, we conclude that gACA19-5addCU and gACA36 are two optimized scaffolds for targeted pseudouridylation experiments in mammalian cells. The gsnoRNAs served as the first-generation RESTART (RESTARTv1). We tested the general applicability of RESTART using four additional cell lines originating from distinct tissues, including three human cell lines and one murine cell line. The H/ACA box snoRNP machinery primarily installs Ψ in rRNA and snRNA and is highly and ubiquitously expressed in various cells and tissues (Figures S2A–S2C). In line with this, efficient PTC-readthrough events were observed for all cell lines using Reporter-3 (Figure S2D). The readthrough efficiency also correlated with the expression level of DKC1 proteins (Figures S1L, S2C, and S2D). Together, these observations suggest that RESTART is a widely applicable tool to suppress PTC in different mammalian systems. We further sought for factors that can improve RNA-editing efficiency of RESTART. There exist two protein-coding DKC1 isoforms in human cells: DKC1-isoform1 gives rise to the canonical form of protein containing bipartite N- and C-terminal nuclear localization signals (NLSs), which is concentrated in nucleoli where rRNA modifications take place; in addition, transcript of the gene can be alternatively spliced to give DKC1-isoform3, which lacks the C-terminal NLS and has cytoplasmic localization (Figures 3A and 3B ).45Angrisani A. Turano M. Paparo L. Di Mauro C. Furia M. A new human dyskerin isoform with cytoplasmic localization.Biochim. Biophys. Acta. 2011; 1810: 1361-1368https://doi.org/10.1016/j.bbagen.2011.07.012Crossref PubMed Scopus (17) Google Scholar DKC1-isoform1 is expressed in multiple tissues, at approximately 20-fold greater levels than that of isoform3 (Figure S2B), consistent with the literature.45Angrisani A. Turano M. Paparo L. Di Mauro C. Furia M. A new human dyskerin isoform with cytoplasmic localization.Biochim. Biophys. Acta. 2011; 1810: 1361-1368https://doi.org/10.1016/j.bbagen.2011.07.012Crossref PubMed Scopus (17) Google Scholar To test whether overexpressed DKC1 may influence RESTART, we generated DKC1-isoform1 and -isoform3 stable overexpression cell lines, respectively, and transfected with Reporter-3 (Figures 3C and 3D). We found that DKC1-isoform1 overexpression slightly increased the relative fraction of EGFP positive cells and intensity (to 1.2- and 1.3-fold compared with that of control cells, respectively) (Figures 3E–3G). Unexpectedly, in DKC1-isoform3 overexpressed cells, the relative fraction of EGFP positive cells and intensity were greatly increased to 2.5- and 5.2- fold, respectively (Figures 3E–3G). These observations were further confirmed using Reporter-1 and by transient overexpression of DKC1 (Figures S2E–S2H). We also generated a series of N-terminal truncations to further delete the NLS of DKC1-isoform3 and found a similar efficiency of PTC readthrough and subcellular localization as full-length isoform3 (Figures S2I and S2J). On the other hand, overexpressing GAR1, NHP2, and NOP10, the other three core proteins of H/ACA snoRNP did not show improvement in PTC readthrough (Figure S2K). With the finding of DKC1-isoform3, we term the combination of gsnoRNAs and overexpressed DKC1-isoform3 as the second-generation RESTART (RESTARTv2). We then performed direct comparison of RESTART with REPAIR.13Cox D.B.T. Gootenberg J.S. Abudayyeh O.O. Franklin B. Kellner M.J. Joung J. Zhang F. RNA editing with CRISPR-Cas13.Science. 2017; 358: 1019-1027https://doi.org/10.11" @default.
• W4311468629 created "2022-12-26" @default.
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• W4311468629 date "2023-01-01" @default.
• W4311468629 modified "2023-10-12" @default.
• W4311468629 title "CRISPR-free, programmable RNA pseudouridylation to suppress premature termination codons" @default.
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