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A programmable system to methylate and demethylate N6-methyladenosine (m6A) on specific RNA transcripts in mammalian cells

  • Chen Chang
    Affiliations
    Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Cancer Centre, Faculty of Health Sciences, University of Macau, Taipa, China
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  • Gang Ma
    Affiliations
    Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
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  • Edwin Cheung
    Correspondence
    For correspondence: Andrew P. Hutchins; Edwin Cheung
    Affiliations
    Cancer Centre, Faculty of Health Sciences, University of Macau, Taipa, China
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  • Andrew P. Hutchins
    Correspondence
    For correspondence: Andrew P. Hutchins; Edwin Cheung
    Affiliations
    Department of Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China

    Shenzhen Key Laboratory of Gene Regulation and Systems Biology, School of Life Sciences, Southern University of Science and Technology, Shenzhen, China
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Open AccessPublished:September 23, 2022DOI:https://doi.org/10.1016/j.jbc.2022.102525
      RNA N6-methyladenosine (m6A) is the most abundant internal mRNA modification and forms part of an epitranscriptomic system that modulates RNA function. m6A is reversibly catalyzed by specific enzymes, and those modifications can be recognized by RNA-binding proteins that in turn regulate biological processes. Although there are many reports demonstrating m6A participation in critical biological functions, this exploration has mainly been conducted through the global KO or knockdown of the writers, erasers, or readers of m6A. Consequently, there is a lack of information about the role of m6A on single transcripts in biological processes, posing a challenge in understanding the biological functions of m6A. Here, we demonstrate a CRISPR/dCas13a-based RNA m6A editors, which can target RNAs using a single or multiple CRISPR RNA array to methylate or demethylate m6A in human 293T cells and mouse embryonic stem cells. We systematically assay its capabilities to enable the targeted rewriting of m6A dynamics, including modulation of circular RNA translation and transcript half-life. Finally, we use the system to specifically modulate m6A levels on the noncoding XIST (X-inactive specific transcript) to modulate X chromosome silencing and activation. The editors described here can be used to explore the roles of m6A in biological processes.

      Keywords

      Abbreviations:

      Cas (CRISPR-associated protein), cDNA (complementary DNA), crRNA (CRISPR RNA), ESC (embryonic stem cell), HEK293T (human embryonic kidney 293T cell line), IP (immunoprecipitation), IRES (internal ribosome entry site), lncRNA (long noncoding RNA), m6A (N6-methyladenosine), MeRIP–qPCR (m6A RNA immunoprecipitation–qPCR), mESC (mouse embryonic stem cell), MTD (methyltransferase domain), qPCR (quantitative PCR), XIST (X-inactive specific transcript)
      Analogous to the DNA epigenetic system, RNA is also chemically modified to impart epitranscriptomic information. N6-methyladenosine (m6A) is the most abundant internal RNA chemical modification in higher eukaryotes and marks all classes of RNA, including coding and noncoding transcripts (
      • Fu Y.
      • Dominissini D.
      • Rechavi G.
      • He C.
      Gene expression regulation mediated through reversible m6A RNA methylation.
      ,
      • Roignant J.Y.
      • Soller M.
      m(6)A in mRNA: an ancient mechanism for fine-tuning gene expression.
      ,
      • Desrosiers R.
      • Friderici K.
      • Rottman F.
      Identification of methylated nucleosides in messenger RNA from novikoff hepatoma cells.
      ). In a typical cell type in mammals, several thousand transcripts are extensively marked by m6A (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ,
      • Dominissini D.
      • Moshitch-Moshkovitz S.
      • Schwartz S.
      • Salmon-Divon M.
      • Ungar L.
      • Osenberg S.
      • et al.
      Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
      ), generally in the 3′UTR in coding transcripts (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ), but anywhere in noncoding transcripts (
      • Coker H.
      • Wei G.
      • Brockdorff N.
      m6A modification of non-coding RNA and the control of mammalian gene expression.
      ). Increasingly, m6A has been implicated in a diverse range of cellular processes, centered on RNA metabolism and processing, including RNA stability, alternative splicing, nuclear export, retrotransposon silencing, and protein translation (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ,
      • Petropoulos S.
      • Edsgard D.
      • Reinius B.
      • Deng Q.
      • Panula S.P.
      • Codeluppi S.
      • et al.
      Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos.
      ,
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Liu J.
      • Dou X.
      • Chen C.
      • Chen C.
      • Liu C.
      • Xu M.M.
      • et al.
      N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.
      ,
      • Mauer J.
      • Luo X.
      • Blanjoie A.
      • Jiao X.
      • Grozhik A.V.
      • Patil D.P.
      • et al.
      Reversible methylation of m(6)Am in the 5' cap controls mRNA stability.
      ,
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ,
      • Yu J.
      • Chen M.
      • Huang H.
      • Zhu J.
      • Song H.
      • Zhu J.
      • et al.
      Dynamic m6A modification regulates local translation of mRNA in axons.
      ,
      • Xiang J.F.
      • Yang Q.
      • Liu C.X.
      • Wu M.
      • Chen L.L.
      • Yang L.
      N(6)-Methyladenosines modulate A-to-I RNA editing.
      ,
      • Barbieri I.
      • Tzelepis K.
      • Pandolfini L.
      • Shi J.
      • Millan-Zambrano G.
      • Robson S.C.
      • et al.
      Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control.
      ,
      • Shi H.
      • Zhang X.
      • Weng Y.L.
      • Lu Z.
      • Liu Y.
      • Lu Z.
      • et al.
      m(6)A facilitates hippocampus-dependent learning and memory through YTHDF1.
      ,
      • Xiao W.
      • Adhikari S.
      • Dahal U.
      • Chen Y.S.
      • Hao Y.J.
      • Sun B.F.
      • et al.
      Nuclear m(6)A reader YTHDC1 regulates mRNA splicing.
      ,
      • Wei J.
      • He C.
      Chromatin and transcriptional regulation by reversible RNA methylation.
      ). In addition, m6A has also been recognized to play a role in several biological processes and diseases, including embryonic stem cell differentiation (
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ,
      • Batista P.J.
      • Molinie B.
      • Wang J.
      • Qu K.
      • Zhang J.
      • Li L.
      • et al.
      m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.
      ), oncogenesis (
      • Barbieri I.
      • Tzelepis K.
      • Pandolfini L.
      • Shi J.
      • Millan-Zambrano G.
      • Robson S.C.
      • et al.
      Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control.
      ,
      • Lin S.
      • Choe J.
      • Du P.
      • Triboulet R.
      • Gregory R.I.
      The m(6)A methyltransferase METTL3 promotes translation in human cancer cells.
      ,
      • Deng X.
      • Su R.
      • Weng H.
      • Huang H.
      • Li Z.
      • Chen J.
      RNA N(6)-methyladenosine modification in cancers: current status and perspectives.
      ,
      • Liu J.
      • Eckert M.A.
      • Harada B.T.
      • Liu S.M.
      • Lu Z.
      • Yu K.
      • et al.
      m(6)A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.
      ,
      • Li Z.
      • Weng H.
      • Su R.
      • Weng X.
      • Zuo Z.
      • Li C.
      • et al.
      FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase.
      ,
      • Li Z.
      • Peng Y.
      • Li J.
      • Chen Z.
      • Chen F.
      • Tu J.
      • et al.
      N6-methyladenosine regulates glycolysis of cancer cells through PDK4.
      ), neurogenesis (
      • Engel M.
      • Eggert C.
      • Kaplick P.M.
      • Eder M.
      • Roh S.
      • Tietze L.
      • et al.
      The role of m(6)A/m-RNA methylation in stress response regulation.
      ,
      • Zhang Z.
      • Wang M.
      • Xie D.
      • Huang Z.
      • Zhang L.
      • Yang Y.
      • et al.
      METTL3-mediated N(6)-methyladenosine mRNA modification enhances long-term memory consolidation.
      ), and X-chromosome inactivation (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ,
      • Chu C.
      • Zhang Q.C.
      • da Rocha S.T.
      • Flynn R.A.
      • Bharadwaj M.
      • Calabrese J.M.
      • et al.
      Systematic discovery of Xist RNA binding proteins.
      ,
      • Huisman B.
      • Manske G.
      • Carney S.
      • Kalantry S.
      Functional dissection of the m6A RNA modification.
      ,
      • Brown C.J.
      • Hendrich B.D.
      • Rupert J.L.
      • Lafrenière R.G.
      • Xing Y.
      • Lawrence J.
      • et al.
      The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus.
      ,
      • Engreitz J.M.
      • Ollikainen N.
      • Guttman M.
      Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression.
      ), amongst many others (
      • Fu Y.
      • Dominissini D.
      • Rechavi G.
      • He C.
      Gene expression regulation mediated through reversible m6A RNA methylation.
      ,
      • Deng X.
      • Su R.
      • Weng H.
      • Huang H.
      • Li Z.
      • Chen J.
      RNA N(6)-methyladenosine modification in cancers: current status and perspectives.
      ,
      • Roundtree I.A.
      • Evans M.E.
      • Pan T.
      • He C.
      Dynamic RNA modifications in gene expression regulation.
      ,
      • Yang Y.
      • Hsu P.J.
      • Chen Y.S.
      • Yang Y.G.
      Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism.
      ). However, these mechanisms have been largely explored through the global KO or knockdown of m6A effectors, such as writers (METTL3/METTL14/WTAP/RBM15/15B), erasers (FTO/ALKBH5), or the readers (YTH protein family) (
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ,
      • Barbieri I.
      • Tzelepis K.
      • Pandolfini L.
      • Shi J.
      • Millan-Zambrano G.
      • Robson S.C.
      • et al.
      Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control.
      ,
      • Xiao W.
      • Adhikari S.
      • Dahal U.
      • Chen Y.S.
      • Hao Y.J.
      • Sun B.F.
      • et al.
      Nuclear m(6)A reader YTHDC1 regulates mRNA splicing.
      ,
      • Li Z.
      • Weng H.
      • Su R.
      • Weng X.
      • Zuo Z.
      • Li C.
      • et al.
      FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase.
      ,
      • Roundtree I.A.
      • Evans M.E.
      • Pan T.
      • He C.
      Dynamic RNA modifications in gene expression regulation.
      ,
      • Zaccara S.
      • Jaffrey S.R.
      A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA.
      ). Considering m6A marks several thousand transcripts in a typical cell type, the scope for pleiotropic effects from these knockdowns/KOs is large. Hence, to reveal the specific functions of m6A on individual transcripts, there is a need to specifically modulate m6A on single or multiple transcripts.
      The CRISPR systems from bacteria or archaea utilize CRISPR RNAs (crRNAs) to guide CRISPR-associated proteins (Cas) specifically cleave invading DNA- and RNA-based pathogens (
      • Terns M.P.
      • Terns R.M.
      CRISPR-based adaptive immune systems.
      ,
      • Bhaya D.
      • Davison M.
      • Barrangou R.
      CRISPR-cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.
      ,
      • Barrangou R.
      • Fremaux C.
      • Deveau H.
      • Richards M.
      • Boyaval P.
      • Moineau S.
      • et al.
      CRISPR provides acquired resistance against viruses in prokaryotes.
      ,
      • Koonin E.V.
      • Makarova K.S.
      • Zhang F.
      Diversity, classification and evolution of CRISPR-Cas systems.
      ). This bacterial immune system has been repurposed to create a powerful suite of genome and epigenome modification tools (
      • Knott G.J.
      • Doudna J.A.
      CRISPR-Cas guides the future of genetic engineering.
      ,
      • Komor A.C.
      • Badran A.H.
      • Liu D.R.
      CRISPR-based Technologies for the manipulation of eukaryotic genomes.
      ,
      • Wright A.V.
      • Nunez J.K.
      • Doudna J.A.
      Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering.
      ,
      • Goell J.H.
      • Hilton I.B.
      CRISPR/Cas-Based epigenome editing: advances, applications, and clinical utility.
      ,
      • Jinek M.
      • Chylinski K.
      • Fonfara I.
      • Hauer M.
      • Doudna J.A.
      • Charpentier E.
      A programmable dual-RNA guided DNA endonuclease in adaptive bacterial immunity.
      ,
      • Cong L.
      • Ran F.A.
      • Cox D.
      • Lin S.
      • Barretto R.
      • Habib N.
      • et al.
      Multiplex genome engineering using CRISPR/Cas systems.
      ,
      • Mali P.
      • Yang L.
      • Esvelt K.M.
      • Aach J.
      • Guell M.
      • DiCarlo J.E.
      • et al.
      RNA-guided human genome engineering via Cas9.
      ,
      • Makarova K.S.
      • Zhang F.
      • Koonin E.V.
      SnapShot: class 1 CRISPR-cas systems.
      ,
      • Makarova K.S.
      • Zhang F.
      • Koonin E.V.
      SnapShot: class 2 CRISPR-cas systems.
      ). Whilst many Cas proteins such as Cas9 target DNA, there are also Cas proteins that target RNA (
      • Wright A.V.
      • Nunez J.K.
      • Doudna J.A.
      Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering.
      ,
      • Makarova K.S.
      • Zhang F.
      • Koonin E.V.
      SnapShot: class 1 CRISPR-cas systems.
      ,
      • Makarova K.S.
      • Zhang F.
      • Koonin E.V.
      SnapShot: class 2 CRISPR-cas systems.
      ). LwaCas13a contains two RNase domains and can degrade RNA guided by a crRNA sequence (
      • Abudayyeh O.O.
      • Gootenberg J.S.
      • Essletzbichler P.
      • Han S.
      • Joung J.
      • Belanto J.J.
      • et al.
      RNA targeting with CRISPR–Cas13.
      ,
      • East-Seletsky A.
      • O'Connell M.R.
      • Knight S.C.
      • Burstein D.
      • Cate J.H.
      • Tjian R.
      • et al.
      Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.
      ,
      • Liu L.
      • Li X.
      • Wang J.
      • Wang M.
      • Chen P.
      • Yin M.
      • et al.
      Two distant catalytic sites are responsible for C2c2 RNase activities.
      ,
      • Abudayyeh O.O.
      • Gootenberg J.S.
      • Konermann S.
      • Joung J.
      • Slaymaker I.M.
      • Cox D.B.
      • et al.
      C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.
      ,
      • Konermann S.
      • Lotfy P.
      • Brideau N.J.
      • Oki J.
      • Shokhirev M.N.
      • Hsu P.D.
      Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.
      ). A catalytic-dead Cas13a was generated (dCas13a), which when guided by a crRNA, can function as an RNA sequence–specific binding protein that can deliver dCas13a-fused proteins to specific RNAs (
      • Abudayyeh O.O.
      • Gootenberg J.S.
      • Essletzbichler P.
      • Han S.
      • Joung J.
      • Belanto J.J.
      • et al.
      RNA targeting with CRISPR–Cas13.
      ,
      • Smargon A.A.
      • Shi Y.J.
      • Yeo G.W.
      RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.
      ,
      • Cox D.B.T.
      • Gootenberg J.S.
      • Abudayyeh O.O.
      • Franklin B.
      • Kellner M.J.
      • Joung J.
      • et al.
      RNA editing with CRISPR-Cas13.
      ). Thus, catalytic-dead dCas13a, fused to RNA-modifying enzymes, cotransfected with specific crRNAs can potentially modulate information on single transcripts. To date, several m6A-editing systems have been characterized, dCas13b-METTL3 (
      • Wilson C.
      • Chen P.J.
      • Miao Z.
      • Liu D.R.
      Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase.
      ), dCas9-METTL3-METTL14 (
      • Liu X.M.
      • Zhou J.
      • Mao Y.
      • Ji Q.
      • Qian S.B.
      Programmable RNA N(6)-methyladenosine editing by CRISPR-Cas9 conjugates.
      ), dCas13b-ALKBH5 (
      • Li J.
      • Chen Z.
      • Chen F.
      • Xie G.
      • Ling Y.
      • Peng Y.
      • et al.
      Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.
      ), and dCas13a-ALKBH5 (
      • Chen X.
      • Zhao Q.
      • Zhao Y.-L.
      • Chai G.-S.
      • Cheng W.
      • Zhao Z.
      • et al.
      Targeted RNA N6-methyladenosine demethylation controls cell fate transition in human pluripotent stem cells.
      ), dCas13b/SunTag (
      • Mo J.
      • Chen Z.
      • Qin S.
      • Li S.
      • Liu C.
      • Zhang L.
      • et al.
      Trades: targeted RNA demethylation by SunTag system.
      ), dCasRx-METTL3/ALKBH5 (
      • Xia Z.
      • Tang M.
      • Ma J.
      • Zhang H.
      • Gimple R.C.
      • Prager B.C.
      • et al.
      Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase.
      ), and a photoactivatable system (
      • Zhao J.
      • Li B.
      • Ma J.
      • Jin W.
      • Ma X.
      Photoactivatable RNA N(6) -methyladenosine editing with CRISPR-cas13.
      ). The advantages and disadvantages of these systems were recently reviewed (
      • Sun X.
      • Wang D.O.
      • Wang J.
      Targeted manipulation of m(6)A RNA modification through CRISPR-Cas-based strategies.
      ).
      Here, we generated dCas13a fused with full-length METTL3 or the core m6A methyltransferase domain (MTD) containing the methylation domain and two core zinc finger domains of METTL3 (
      • Huang J.
      • Dong X.
      • Gong Z.
      • Qin L.Y.
      • Yang S.
      • Zhu Y.L.
      • et al.
      Solution structure of the RNA recognition domain of METTL3-METTL14 N(6)-methyladenosine methyltransferase.
      ,
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.
      • Zhang D.
      • Liu Z.
      • et al.
      Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.
      ,
      • Śledź P.
      • Jinek M.
      Structural insights into the molecular mechanism of the m(6)A writer complex.
      ,
      • Wang P.
      • Doxtader Katelyn A.
      • Nam Y.
      Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases.
      ) or with the m6A demethylase FTO (
      • Fu Y.
      • Jia G.
      • Pang X.
      • Wang R.N.
      • Wang X.
      • Li C.J.
      • et al.
      FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
      ,
      • Han Z.
      • Niu T.
      • Chang J.
      • Lei X.
      • Zhao M.
      • Wang Q.
      • et al.
      Crystal structure of the FTO protein reveals basis for its substrate specificity.
      ). The dCas13a fusions are available in vectors compatible with liposomal transfection or lentiviral vectors suitable for hard-to-transfect cells. The system works in human and mouse cells. We show that our CRISPR/dCas13a-associated RNA m6A-editing system can be specifically navigated by crRNAs to modulate m6A on individual coding or noncoding RNAs. We demonstrate the efficacy of this system to modulate circular RNA translational efficiency and RNA transcript half-life. In addition, by taking advantage of the intrinsic capability for Cas13a to mature pre-crRNA, we show how the dCas13a fusions can process a programmable crRNA array containing multiple crRNAs to target several transcripts simultaneously for m6A editing. Finally, we utilize this system to edit m6A on the long noncoding RNA (lncRNA) XIST (X-inactive specific transcript), resulting in the reactivation of XIST-repressed genes, or active silencing in METTL3 knockdown cells. This work describes a series of tools that can edit m6A in multiple mammalian cellular contexts on specific RNAs to explore the effect of m6A on transcript activity.

      Results

      Construction of dCas13a-associated m6A-editing systems in mammalian cells

      m6A RNA methylation is catalyzed by several protein complexes containing multiple protein subunits, for example, METTL3, METTL14, WTAP, and so on (
      • Yang Y.
      • Hsu P.J.
      • Chen Y.S.
      • Yang Y.G.
      Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism.
      ,
      • Huang J.
      • Dong X.
      • Gong Z.
      • Qin L.Y.
      • Yang S.
      • Zhu Y.L.
      • et al.
      Solution structure of the RNA recognition domain of METTL3-METTL14 N(6)-methyladenosine methyltransferase.
      ,
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.
      • Zhang D.
      • Liu Z.
      • et al.
      Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.
      ,
      • Zhang X.
      • Wei L.H.
      • Wang Y.
      • Xiao Y.
      • Liu J.
      • Zhang W.
      • et al.
      Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.
      ,
      • Zheng G.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      ,
      • Xu C.
      • Liu K.
      • Tempel W.
      • Demetriades M.
      • Aik W.
      • Schofield C.J.
      • et al.
      Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation.
      ,
      • Feng C.
      • Liu Y.
      • Wang G.
      • Deng Z.
      • Zhang Q.
      • Wu W.
      • et al.
      Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.
      ). However, amongst these proteins, METTL3 is the catalytic enzyme (
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.
      • Zhang D.
      • Liu Z.
      • et al.
      Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.
      ,
      • Wang P.
      • Doxtader Katelyn A.
      • Nam Y.
      Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases.
      ). m6A demethylation can be accomplished by a single catalytic enzyme (
      • Huang J.
      • Dong X.
      • Gong Z.
      • Qin L.Y.
      • Yang S.
      • Zhu Y.L.
      • et al.
      Solution structure of the RNA recognition domain of METTL3-METTL14 N(6)-methyladenosine methyltransferase.
      ,
      • Han Z.
      • Niu T.
      • Chang J.
      • Lei X.
      • Zhao M.
      • Wang Q.
      • et al.
      Crystal structure of the FTO protein reveals basis for its substrate specificity.
      ,
      • Zheng G.
      • Dahl J.A.
      • Niu Y.
      • Fedorcsak P.
      • Huang C.M.
      • Li C.J.
      • et al.
      ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
      ,
      • Feng C.
      • Liu Y.
      • Wang G.
      • Deng Z.
      • Zhang Q.
      • Wu W.
      • et al.
      Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.
      ), such as FTO or ALKBH5. Hence, we selected human METTL3 full-length protein, or a truncated methylation domain (MTD), containing the methylation domain and two core zinc finger domains, as m6A writers, and FTO as an m6A eraser.
      To construct an m6A-editing system, we fused the catalytically inactivated LwaCas13a (dCas13a) (
      • Abudayyeh O.O.
      • Gootenberg J.S.
      • Essletzbichler P.
      • Han S.
      • Joung J.
      • Belanto J.J.
      • et al.
      RNA targeting with CRISPR–Cas13.
      ,
      • East-Seletsky A.
      • O'Connell M.R.
      • Knight S.C.
      • Burstein D.
      • Cate J.H.
      • Tjian R.
      • et al.
      Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.
      ) protein with METTL3/MTD or FTO (Figs. 1A and S1A). As controls, we constructed catalytic-dead mutants of METTL3D395A (
      • Wang X.
      • Feng J.
      • Xue Y.
      • Guan Z.
      • Zhang D.
      • Liu Z.
      • et al.
      Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.
      ,
      • Wang P.
      • Doxtader Katelyn A.
      • Nam Y.
      Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases.
      ) and FTOY108A (
      • Han Z.
      • Niu T.
      • Chang J.
      • Lei X.
      • Zhao M.
      • Wang Q.
      • et al.
      Crystal structure of the FTO protein reveals basis for its substrate specificity.
      ) fused to dCas13a (Fig. S1A). All these fusion systems contain an N-terminal dCas13a, followed by a 13 amino acid GGS linker, a hemagglutinin tag, three nuclear localization signals, and a P2A (self-cleaving peptide sequence) fused with mCherry to report transfection efficiency (Fig. S1A). The crRNA is contained on a second U6-promoter–containing vector, which is cotransfected along with the fusion protein (Fig. S1A). After transfection of the editing systems into 293T cells, mCherry fluorescence was detected and transfection efficiency was estimated as 62% (Fig. S1, BE). Importantly, transfection of dCas13a-METTL3 or dCas13a-FTO did neither substantially alter the cell cycle nor increase the proportion of dead cells (Fig. S1, FH), indicating the transgenes were not inducing cellular toxicity. Together, these vectors make up an m6A-editing system that can be targeted by the crRNA to a specific transcript.
      Figure thumbnail gr1
      Figure 1A programmable m6A methylation and demethylation system. A, schematic of the m6A-editing systems. B, Western blot of GFP or βACTIN from 293T cells transfected with crNT (nontargeting crRNA) or crRNAs targeting specific sites around the ATG codon of the circular nonDRACH-containing plasmid (D), and dCas13a-METTL3, or the catalytic-dead dCas13a-METTL3D395A. This experiment was repeated three times with similar results. Molecular weight markers (in kilodalton) are indicated on the left side for this and all subsequent Western blots. C, Western blot of GFP or βACTIN from 293T cells transfected with crNT or crRNAs targeting specific sites around the ATG codon of the 2-DRACH-containing circular plasmid (D) and dCas13a-FTO or the catalytic-dead dCas13a-FTOY108A. This experiment was repeated three times with similar results. D, SELECT assay of m6A level on nonDRACH circGFP at position 22 (A), in cells transfected with dCas13a-METTL3, the catalytic-null catalytic-dead dCas13a-METTL3D395A, and crRNAs targeting the indicated base pairs relative to the GFPs ATG. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-METTL3/crNT sample. Significance is from a two-tailed unpaired Student’s t test, for this and all statistical tests unless otherwise stated. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. E, SELECT assay of m6A level on 2DRACH circGFP at position 4 (A), in cells transfected with dCas13a-FTO, the catalytic-null catalytic-dead dCas13a-FTOY108A, and crRNAs targeting the indicated base pairs relative to the GFPs ATG. Y-axis indicates inversed normalized m6A abundance normalized to dCas13a-FTO/crNT sample. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. F, whole transcriptome m6A measurement for RNA m6A level in 293T cells transfected with the indicated crRNAs and dCas13a-METTL3, catalytic-dead dCas13a-METTL3D395A. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. p Value is from a one-way ANOVA. G, whole transcriptome m6A measurement for RNA m6A level in293T cells transfected with the indicated crRNAs and dCas13a-FTO or catalytic-dead dCas13a-FTOY108A. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. p Value is from a one-way ANOVA. m6A, N6-methyladenosine.

      Manipulating m6A on exogenous circular RNAs modulates their translation

      To explore the activity of the dCas13a fusions, we initially constructed a reporter system to indirectly measure the m6A levels of a specific RNA. To this end, we exploited the ability of m6A to promote protein translation efficiency from circular RNAs in human cells (
      • Liang D.
      • Wilusz J.E.
      Short intronic repeat sequences facilitate circular RNA production.
      ,
      • Wang Y.
      • Wang Z.
      Efficient backsplicing produces translatable circular mRNAs.
      ,
      • Yang Y.
      • Fan X.
      • Mao M.
      • Song X.
      • Wu P.
      • Zhang Y.
      • et al.
      Extensive translation of circular RNAs driven by N(6)-methyladenosine.
      ). To determine if our system can modify specific m6A sites on RNA, we generated a circular RNA expression system to serve as an indirect readout of m6A (Fig. S2A) (
      • Liang D.
      • Wilusz J.E.
      Short intronic repeat sequences facilitate circular RNA production.
      ,
      • Yang Y.
      • Fan X.
      • Mao M.
      • Song X.
      • Wu P.
      • Zhang Y.
      • et al.
      Extensive translation of circular RNAs driven by N(6)-methyladenosine.
      ). We generated an artificial circular RNA expression system where the coding sequence of GFP was separated linearly into two parts between an IRES (internal ribosome entry site) to form an “FP-IRES-(ATG)G” linear sequence that would back-splice to form a complete circular IRES-GFP coding sequence (Fig. S2A). Based on several reports (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ,
      • Dominissini D.
      • Moshitch-Moshkovitz S.
      • Schwartz S.
      • Salmon-Divon M.
      • Ungar L.
      • Osenberg S.
      • et al.
      Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
      ,
      • Linder B.
      • Grozhik A.V.
      • Olarerin-George A.O.
      • Meydan C.
      • Mason C.E.
      • Jaffrey S.R.
      Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.
      ), the METTL3–METTL14–WTAP complex prefers to methylate the A in a DRACH consensus motif, where D donates A, G, or U, R donates A or G, and H donates A, C, or U nucleotides (Fig. S2B). Hence, we generated a series of reporters that include 2, 1, and 0 GGACU sequences or no A nucleotides (Fig. S2C). In the endogenous METTL3–METTL14 complex, METTL14 targets METTL3 to DRACH motifs (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ,
      • Dominissini D.
      • Moshitch-Moshkovitz S.
      • Schwartz S.
      • Salmon-Divon M.
      • Ungar L.
      • Osenberg S.
      • et al.
      Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
      ,
      • Linder B.
      • Grozhik A.V.
      • Olarerin-George A.O.
      • Meydan C.
      • Mason C.E.
      • Jaffrey S.R.
      Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.
      ); however, as our system does not use METTL14, the requirement for DRACH targeting is relaxed, and potentially any available A nucleotide is a target. Consequently, we used the no DRACH (low levels of translation) and the 2-DRACH (high levels of translation) circular reporters as an indirect readout for changes in m6A levels (Fig. S2, B and C).
      Several sites were selected as targets for a crRNA, including locations just outside the IRES region, and before or after the start codon (defined as position 1) of the GFP. crRNAs were constructed targeting specific sequences (Fig. S2D and Table S1). We then cotransfected a crRNA with dCas13a-METTL3 with the no-DRACH reporter. In a separate experiment, we used dCas13a-FTO cotransfected with the 2-DRACH plasmid. We assayed the indirect impact of m6A on protein translation by measuring the level of GFP in a Western blot. Western blot showed that, compared with nontargeting crRNA, crRNAs targeting the circular RNA resulted in improved GFP translation from the dCas13a-METTL3 transfected cells and impaired translation when using the dCas13a-FTO system (Figs. 1, B and C and S3, A and B). Interestingly, GFP expression was affected even when the crRNA was up to 107 nucleotides 5′ of the ATG, in the case of dCas13a-METTL3 (Fig. S3C) or up to 95 nucleotides 5′ of the ATG, in the case of dCas13a-FTO (Fig. S3D). We then performed an assay based on competitive elongation and ligation-based quantitative PCR (qPCR) to directly measure the level of m6A at a specific nucleotide (“SELECT” assay) (
      • Xiao Y.
      • Wang Y.
      • Tang Q.
      • Wei L.
      • Zhang X.
      • Jia G.
      An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6) -methyladenosine modification.
      ). In the SELECT qPCR-based assay, the presence of an m6A impairs the production of the complementary DNA (cDNA) product. Increased m6A manifests as a decreased cycle threshold, whereas decreased m6A increases the cycle threshold (
      • Xiao Y.
      • Wang Y.
      • Tang Q.
      • Wei L.
      • Zhang X.
      • Jia G.
      An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6) -methyladenosine modification.
      ). SELECT assay showed that dCas13a-METTL3 with specific crRNAs resulted in upregulation of m6A, which was not present when using the catalytic mutant form of METTL3D395A (Fig. 1D). Conversely, dCas13a-FTO decreased m6A levels, whereas dCas13a-FTOY108A catalytic mutant did not change m6A levels (Fig. 1E). One concern is that the overexpression of dCas13a-METTL3/FTO may act as a dominant positive and nonspecifically methylate RNA. Assay for the transcriptome-wide level of m6A indicated that the dCas13a fusions did not significantly change the transcriptome level of m6A (Fig. 1, F and G). We also confirmed that the dCas13a-METTL3/FTO constructs did not alter endogenous METTL3 or FTO protein levels (Fig. S3, E and F), and the dCas fusions were only expressed at around 20% of the level of the endogenous protein (Fig. S3, G and H). Finally, there was no effect on endogenous METTL3 when dCas13a-FTO was expressed nor on the inverse (Fig. S3, I and J).

      dCas13a-METTL3, dCas13a-MTD, and dCas13a-FTO fusions can modulate m6A on endogenous transcripts and alter their half-life in human embryonic kidney 293T cells

      We next set out to use the dCas13a fusions on endogenous transcripts. To verify whether the dCas13a fusions can edit m6A on endogenous transcripts, we chose several candidate RNAs expressed in human embryonic kidney 293T (HEK293T) cells, including mRNAs (H1F0, SGK1, and ID3) and an lncRNA (MALAT1). Reanalysis of m6A sequencing data in 293T cells indicated these transcripts are expressed and marked by m6A (Fig. S4, AD) (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ,
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Zaccara S.
      • Jaffrey S.R.
      A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA.
      ,
      • Fu Y.
      • Zhuang X.
      m6A-binding YTHDF proteins promote stress granule formation.
      ). We next transfected cells with the dCas13a fusions and crRNAs. SELECT assay indicated that m6A levels of H1F0 and SGK1 were significantly increased in the dCas13a-MTD but were unaltered with the nontargeting crRNA or with the MTD catalytic mutant (Fig. 2A). dCas13a-FTO with the same crRNAs against H1F0 and SGK1 led to a significant decrease in m6A levels (Fig. 2B). m6A RNA immunoprecipitation (IP)–qPCR (MeRIP–qPCR) supported the results from SELECT, and indicated dCas13a-MTD resulted in significantly increased m6A levels on H1F0 transcripts (Fig. 2C), whereas dCas13a-FTO led to a significant decrease in m6A on H1F0 or ID3 transcripts when the respective crRNAs were cotransfected (Fig. 2D).
      Figure thumbnail gr2
      Figure 2m6A editors can alter the half-life of specific endogenous transcripts. A, SELECT assay for m6A level on SGK1 and H1F0, with dCas13a-MTD and catalytic-null and crRNA targeting SGK1 or H1F0. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-MTD/crNT sample. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. B, SELECT assay for m6A level on SGK1 and H1F0, with dCas13a-FTO and catalytic-null and crRNA targeting SGK1 or H1F0. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-FTO/crNT sample. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. C, MeRIP–qPCR assay in 293T cells with dCas13a-MTD cotransfected with crRNA against H1F0. Data were normalized to the dCas13a-MTD/crNT sample and against the input. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. D, MeRIP–qPCR assay in 293T cells with dCas13a-FTO cotransfected with crRNAs against H1F0 or ID3. Data were normalized to the dCas13a-FTO/crNT sample and against the input. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. E, RT–qPCR of METTL3 in 293T cells transfected with shLUC (control shRNA against luciferase) or shMETTL3#1 and shMETTL3#2. Data were normalized to the shLUC sample and ACTB. F, Western blot of METTL3 and βACTIN in 293T cells transfected with shLUC or shMETTL3#1 and shMETTL3#2. G, SGK1 and H1F0 half-life assay, with dCas13a-MTD or the catalytic-null cotransfected with control crNT or crRNA against SGK1 or H1F0. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. 293T cells transfected with an shLUC (luciferase control), shMETTL3#1, or shMETTL3#2 are shown for comparison. Data were normalized to their respective 0 h time points. The right-hand bar charts show the calculated transcript half-lives. H, SGK1 and H1F0 half-life assay, with dCas13a-FTO or the catalytic-null cotransfected with control crNT or crRNA against SGK1 or H1F0. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. 293T cells transfected with an shLUC (luciferase control) shMETTL3#1 or shMETTL3#2 are shown for comparison. Data were normalized to their respective 0 h time points. The right-hand bar charts show the calculated transcript half-lives. crRNA, CRISPR RNA; m6A, N6-methyladenosine; MeRIP–qPCR, m6A RNA immunoprecipitation–qPCR; MTD, methyltransferase domain; qPCR, quantitative PCR.
      We next explored m6A on the lncRNA MALAT1. Using crRNAs specific to MALAT1, transfection of dCas13a-MTD significantly increased m6A levels, as measured by both SELECT and MeRIP–qPCR (Fig. S5, A and B), whereas dCas13a-FTO significantly reduced m6A levels (Fig. S5, C and D). Importantly, SELECT indicated that these changes in m6A levels were only achieved when using catalytically active dCas13a-MTD/METTL3/FTO, as the catalytic mutants did not affect m6A levels (Fig. S5, AD). We confirmed that the dCas13a-MTD/FTO fusions were not acting as dominant positives, as there were no significant changes in the whole transcriptome level of m6A (Fig. S5, E and F). We also explored some off-target effects and used SELECT assay to show there was no off-target methylation/demethylation of MALAT1 when a crRNA against SGK1 of H1F0 was cotransfected with dCas13a-MTD or dCas13a-FTO (Fig. S5, G and H). These data indicate that there is no general modulation of m6A on RNAs, although we cannot rule out off-target effects on other RNAs.
      When m6A writers or readers knocked down or knocked out RNA, half-life has been reported to increase (
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ,
      • Zaccara S.
      • Jaffrey S.R.
      A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA.
      ). However, these results have been demonstrated mainly in global KO or knockdowns of m6A effectors. We wondered if our system could be used to alter the half-life of specific transcripts by changing the level of m6A on target RNAs. First, we validated two shRNAs to knock down METTL3 in 293T cells (
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Lin S.
      • Choe J.
      • Du P.
      • Triboulet R.
      • Gregory R.I.
      The m(6)A methyltransferase METTL3 promotes translation in human cancer cells.
      ). Both shRNAs efficiently depleted both the mRNA and protein levels of METTL3 (Fig. 2, E and F). There was no change in cell morphology or the number of cells dying (Fig. S5I). However, there was an increase in the percentage of G2/M phase cells in the METTL3 knockdowns, suggesting METTL3 is involved in regulating cell cycle–related transcripts. Cells were treated with actinomycin D to block transcription, and RNA abundance was measured by qRT–PCR (
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ). For METTL3 knockdown in 293T cells, we focused on two RNAs, SGK1 and H1F0, as their half-life has been previously characterized as influenced by m6A levels (
      • Zaccara S.
      • Jaffrey S.R.
      A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA.
      ). The results showed that dCas13a-MTD, in cells with METTL3 knockdown, significantly decreased the stability of SGK1 and H1F0 and reduced their half-lives close to WT levels (Fig. 2G). Conversely, dCas13a-FTO, with crRNAs against SGK1 or H1F0, significantly increased the respective mRNA half-lives in WT cells and brought them close to the corresponding transcript half-life in the METTL3 knockdown cells (Fig. 2H). This demonstrates a direct effect of m6A on the half-life of specific transcripts.

      dCas13a-METTL3 and dCas13a-FTO fusions can alter mouse embryonic stem cell–specific RNA half-lives by modulating m6A on endogenous transcripts in mouse embryonic stem cells

      Several research groups have demonstrated that m6A plays a critical role in regulating mouse embryonic stem cell (mESC) fate transitions (
      • Fu Y.
      • Dominissini D.
      • Rechavi G.
      • He C.
      Gene expression regulation mediated through reversible m6A RNA methylation.
      ,
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ,
      • Batista P.J.
      • Molinie B.
      • Wang J.
      • Qu K.
      • Zhang J.
      • Li L.
      • et al.
      m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.
      ,
      • Yang Y.
      • Hsu P.J.
      • Chen Y.S.
      • Yang Y.G.
      Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism.
      ,
      • Ji P.
      • Wang X.
      • Xie N.
      • Li Y.
      N6-Methyladenosine in RNA and DNA: an epitranscriptomic and epigenetic player implicated in determination of stem cell fate.
      ). mESCs are resistant to transfection with liposomal techniques; hence, we generated dCas13a fusions in combined crRNA lentiviral vectors (Fig. S6A). These vectors are one-component vectors containing both the fusion protein and the crRNA. The lentiviral vector is suitable for difficult-to-transfect cell lines, such as mouse and human embryonic stem cells (ESCs), and when transfected into mESCs, mCherry was detected (Fig. 3A). We utilized Mettl3 KO mESCs grown under naïve “2iLIF” (medium including serum, PD98059, CHIR99021, and leukemia inhibitory factor) conditions (
      • Geula S.
      • Moshitch-Moshkovitz S.
      • Dominissini D.
      • Mansour A.A.
      • Kol N.
      • Salmon-Divon M.
      • et al.
      Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
      ) (Fig. S6B) and designed crRNAs targeting two RNAs expressed at high levels in mESCs that are marked by m6A: Klf4 and Sox2 (Fig. S6, C and D). We confirmed that KO of Mettl3 led to increased RNA half-lives of Klf4 and Sox2 compared with WT cells (Fig. S6, E and F). SELECT assay showed that expression of dCas13a-METTL3 led to significantly increased m6A levels on Klf4 and Sox2 mRNAs when the corresponding crRNA was present (Fig. 3, B and C). This effect was lost if the dCas13a-METTL3D395A catalytic mutant was used (Fig. 3, B and C). dCas13a-FTO, conversely, led to decreased m6A levels, and the dCas13a-FTOY108A catalytic-null FTO had no effect on m6A (Fig. 3, D and E). Next, we measured the effect of the dCas13a fusions on RNA half-lives. In Mettl3 KO mESCs when dCas13a-METTL3 was transfected with the corresponding crRNA, the half-lives of Klf4 and Sox2 were significantly shorter compared with the Mettl3 KO cells or crRNA control (crNT) or catalytic-null dCas13a-METTL3D395A (Fig. 3F). Conversely, dCas13a-FTO mediated the opposite effect, and the half-lives of Klf4 and Sox2 significantly increased, although they did not reach the half-lives in the Mettl3 KO mESCs, suggesting dCas13a-FTO does not completely demethylate the transcripts (Fig. 3G). These results show that the dCas13a-METTL3 and dCas13a-FTO lentiviral systems can modulate m6A levels and transcript half-lives in mESCs.
      Figure thumbnail gr3
      Figure 3m6A editors can alter the m6A level and half-life of specific transcripts in mESCs. A, fluorescence microscope images of mESCs transfected with dCas13a-METTL3 or dCas13a-FTO showing mCherry and brightfield views. The scale bar represents 200 μm. B, SELECT assay for m6A level on Klf4, with dCas13a-METTL3 or dCas13a-METTL3D395A catalytic null with crRNA targeting Klf4 or a control nontargeting (crNT) crRNA. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-METTL3/crNT sample. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. C, SELECT assay for m6A level on Sox2, with dCas13a-METTL3 or dCas13a-METTL3D395A catalytic null with crRNA targeting Sox2 or a control crNT crRNA. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-METTL3/crNT sample. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. D, SELECT assay for m6A level on Klf4, with dCas13a-FTO or dCas13a-FTOY108A catalytic null with crRNA targeting Klf4 or a control crNT crRNA. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-FTO/crNT sample. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. E, SELECT assay for m6A level on Sox2, with dCas13a-FTO or dCas13a-FTOY108A catalytic null with crRNA targeting Sox2 or a control crNT crRNA. Y-axis indicates inverse normalized m6A abundance normalized to dCas13a-FTO/crNT sample. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. F, Klf4 and Sox2 half-life assay, with dCas13a-METTL3 or its catalytic null cotransfected with a control crRNA or crRNA against Klf4 or Sox2 into Mettl3 KO cells. Data from WT and KO Mettl3 mESCs are shown for comparison. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. Data were normalized to their respective 0 h time points. The right-hand bar charts show the calculated transcript half-life. G, Klf4 and Sox2 half-life assay, with dCas13a-FTO or its catalytic null cotransfected with a control crRNA or crRNA against Klf4 or Sox2 into WT cells. Data from WT and KO Mettl3 mESCs are shown for comparison. Data are represented as the SEM, n = 3 biological replicates with three technical replicates each. Data were normalized to their respective 0 h time points. The right-hand bar charts show the calculated transcript half-life. crRNA, CRISPR RNA; m6A, N6-methyladenosine; mESC, mouse embryonic stem cell.

      X chromosome-suppressed genes can be reactivated or resilenced by modulating m6A on XIST

      In somatic female cells, only one X chromosome is active. The inactivated X chromosome is silenced through the activity of the noncoding RNA XIST, which coats one of the two chromosomes and recruits polycomb proteins and other epigenetic repressors (
      • Sun L.
      • Fu X.
      • Ma G.
      • Hutchins A.P.
      Chromatin and epigenetic rearrangements in embryonic stem cell fate transitions.
      ,
      • Panning B.
      • Dausman J.
      • Jaenisch R.
      X chromosome inactivation is mediated by Xist RNA stabilization.
      ,
      • Penny G.D.
      • Kay G.F.
      • Sheardown S.A.
      • Rastan S.
      • Brockdorff N.
      Requirement for Xist in X chromosome inactivation.
      ,
      • Sheardown S.A.
      • Duthie S.M.
      • Johnston C.M.
      • Newall A.E.T.
      • Formstone E.J.
      • Arkell R.M.
      • et al.
      Stabilization of Xist RNA mediates initiation of X chromosome inactivation.
      ,
      • Hernández-Muñoz I.
      • Lund A.H.
      • van der Stoop P.
      • Boutsma E.
      • Muijrers I.
      • Verhoeven E.
      • et al.
      Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
      ,
      • Jones A.N.
      • Tikhaia E.
      • Mourão A.
      • Sattler M.
      Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1.
      ). The XIST lncRNA is heavily m6A methylated, and when m6A readers were knocked down, X chromosome genes were reactivated (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ,
      • Chu C.
      • Zhang Q.C.
      • da Rocha S.T.
      • Flynn R.A.
      • Bharadwaj M.
      • Calabrese J.M.
      • et al.
      Systematic discovery of Xist RNA binding proteins.
      ,
      • Huisman B.
      • Manske G.
      • Carney S.
      • Kalantry S.
      Functional dissection of the m6A RNA modification.
      ,
      • Yang Y.
      • Hsu P.J.
      • Chen Y.S.
      • Yang Y.G.
      Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism.
      ). These results suggest that m6A plays a critical role in X chromosome inactivation. However, as m6A marks many thousands of transcripts, the role of m6A specifically on XIST and whether it is a determinant for X chromosome silencing is unclear.
      HEK293T cells were derived from a human embryonic kidney. However, prolonged in vitro cell culture has led to the presence of multiple X chromosomes (
      • Lin Y.-C.
      • Boone M.
      • Meuris L.
      • Lemmens I.
      • Van Roy N.
      • Soete A.
      • et al.
      Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations.
      ), of which all are silenced except one active X (
      • Hernández-Muñoz I.
      • Lund A.H.
      • van der Stoop P.
      • Boutsma E.
      • Muijrers I.
      • Verhoeven E.
      • et al.
      Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
      ). Consequently, they can serve as a model for X chromosome silencing (
      • Hernández-Muñoz I.
      • Lund A.H.
      • van der Stoop P.
      • Boutsma E.
      • Muijrers I.
      • Verhoeven E.
      • et al.
      Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
      ). When we knocked down METTL3 in 293T cells (Fig. 2, E and F), as previously reported, two X chromosome genes GPC4 and ATRX were upregulated (Fig. 4, A and B) (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ). XIST is marked by m6A on several sites of the linear RNA (Fig. 4C). Potentially, not all m6A on XIST is functional. Hence, we designed several crRNAs targeting three sites distributed along the length of the XIST transcript (crXIST#1–#3; Fig. 4C). We then cotransfected crRNAs targeting XIST with dCas13a-MTD in METTL3 knockdown 293T cells to observe if the m6A methylation of XIST leads to suppression of X chromosome genes. RT–qPCR results showed that both ATRX and GPC4 were significantly downregulated by dCas13a-MTD cotransfected with crRNAs targeting XIST (Fig. 4D). This suggests that m6A methylation of XIST RNA leads to suppression of these genes. SELECT assay using primers specific to the location of crXIST#1 showed significantly increased m6A methylation (Fig. 4E). Interestingly, in cells with crXIST#2 or #3, there was no significant change in the methylation at the site of crXIST#1 (Fig. 4E). This suggests that the range of dCas13a-MTD is relatively limited, and this contrasts with the effect seen in circular RNAs where the dCas13a-METTL3/MTD was effective at least 70 nucleotides away from the specific m6A measured in the SELECT assay (Fig. 1, D and E).
      Figure thumbnail gr4
      Figure 4m6A editors can modulate XIST function and reactivate X chromosome-inactivated genes. A, RT–qPCR of the X chromosome gene GPC4 when METTL3 was knocked down in 293T cells. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. Data are normalized to the shLUC sample and ACTB. B, RT–qPCR of the X chromosome gene ATRX when METTL3 was knocked down in 293T cells. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. Data are normalized to the shLUC sample and ACTB. C, genome view (hg38 genome assembly) of m6A RIP-Seq data in 293T cells at the XIST locus. The locations of the crRNAs are indicated with a dotted box. Red indicates m6A enrichment data, and gray tracks indicate the corresponding input data. Transcripts are from GENCODE, version 32. m6A abundance data are from GSE129979 (
      • Wang Y.
      • Xiao Y.
      • Dong S.
      • Yu Q.
      • Jia G.
      Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
      ) (top two rows) or GSE29714 (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ) (bottom two rows). D, RT–qPCR of GPC4 or ATRX when METTL3 was knocked down, compared with cells cotransfected with dCas13a-MTD and crRNAs targeting one or several regions of XIST. Dots indicate the mean for each biological replicate, n = 3 biological replicates with three technical replicates each. Data are normalized to the shMETTL3#1 sample and ACTB. E, SELECT assay for m6A levels at the crXIST#1-targeting site on XIST, when METTL3 was knocked down, compared with cells with dCas13a-MTD and crRNAs targeting one or several regions of XIST. Y-axis indicates inverse normalized m6A abundance normalized to the shLUC sample. Dots indicate the mean for each biological replicate, n = 3 biological replicates with three technical replicates each. F, RT–qPCR of GPC4 or ATRX when METTL3 was knocked down, compared with cells cotransfected with dCas13a- FTO and crRNAs targeting one or several regions of XIST. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. Data are normalized to the shMETTL3#1 sample and ACTB. G, SELECT assay for m6A level at the crXIST#1-targeting site on XIST, when METTL3 was knocked down, compared with shLUC transfected cells with dCas13a-FTO and crRNAs targeting one or several regions of XIST. Y-axis indicates inverse normalized m6A abundance normalized to the shLUC sample. Dots indicate the mean for each biological replicate, n = 3 biological replicates with three technical replicates each. crRNA, CRISPR RNA; m6A, N6-methyladenosine; qPCR, quantitative PCR; RIP-Seq, RNA immunoprecipitation sequencing; XIST, X-inactive specific transcript.
      We next performed the same experiments but using dCas13a-FTO to detect X chromosome activation in WT cells. Transfection of the dCas13a-FTO/crXISTs into 293T cells led to a modest (but significant) upregulation of GPC4 and ATRX expression (Fig. 4F). Presumably, demethylating XIST can only partially overcome epigenetic repression of the X chromosome. Interestingly, in an effect the same as dCas13a-MTD, the dCas13a-FTO/crXIST#1 combination led to demethylation of m6A specifically at the crXIST#1 site, but other crRNAs did not alter methylation at the crXIST#1 site (Fig. 4G). This indicates that on the linear XIST RNA, both dCas13a-MTD and dCas13a-FTO can modulate methylation over a limited range close to the crRNA targeting region. Overall, these results indicate that dCas13a-MTD and dCas13a-FTO can specifically alter m6A on XIST and lead to a change in the expression of X chromosome-associated genes.

      System for editing m6A on multiple RNAs

      As we have shown with the aforementioned XIST experiments, many RNAs have multiple enriched sites of m6A, and potentially several sites must be simultaneously targeted to achieve a biological effect. In addition, genes often have multiple transcript isoforms that can have divergent m6A patterns; hence, it would be useful to modulate m6A at several sites in a single transcript or multiple different genes “transcripts” simultaneously. In Cas13a, the two RNase domains and the pre-crRNA maturation domain are in two distant sites of Cas13a (
      • East-Seletsky A.
      • O'Connell M.R.
      • Knight S.C.
      • Burstein D.
      • Cate J.H.
      • Tjian R.
      • et al.
      Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.
      ,
      • Liu L.
      • Li X.
      • Wang J.
      • Wang M.
      • Chen P.
      • Yin M.
      • et al.
      Two distant catalytic sites are responsible for C2c2 RNase activities.
      ). Hence, we reasoned that dCas13a is still capable of maturing a pre-crRNA array to multiple crRNAs. To investigate whether dCas13a can mature a pre-crRNA array to produce functional crRNAs, we constructed a crRNA array containing multiple crRNAs driven by a single U6 promoter (Fig. 5A). Coexpression of this vector in 293T cells with a crRNA array containing pre-crRNAs against MALAT1, ID3, and H1F0 led to a significant increase in m6A level on all transcripts, as measured by SELECT assay, and the effect was not present if the catalytic-null dCas13a-MTDD395A was used (Fig. 5B). dCas13a-FTO was also capable of processing the crRNA array and could simultaneously significantly decrease the level of m6A in all three transcripts (Fig. 5C). We also compared m6A editing efficiencies between the crRNA array and a cocktail of three single crRNAs. SELECT assay showed that the crRNA array was comparable to the efficiency achieved with mixed single crRNA vectors (Fig. 5, D and E). However, the multiple crRNA array has the advantage of a simplified transfection system, and a second advantage is that pooled crRNA vectors can transfect different cells randomly, whereas the crRNA array means that all crRNAs are present in the same cell. Whilst unlikely to be a problem in cell lines that have high transfection efficiency, in cell lines with poor transfection efficiency, it is helpful to guarantee that transfected cells will receive all crRNAs.
      Figure thumbnail gr5
      Figure 5dCas13a can process a multiple crRNA cassette for targeting multiple transcripts. A, schematic of a multiple crRNA cassette with crRNAs targeting three separate transcripts indicated by the green, red, and blue boxes. The CRISPR array is transcribed to form a pre-crRNA array, containing multiple crRNA scaffolds and the three crRNAs in serial. dCas13a fusions can then mature the pre-crRNAs inside the array to produce multiple mature crRNAs as the maturation domains of dCas13a are intact and functional. B, SELECT assay for inverse normalized m6A abundance of the three genes in the crRNA array, cotransfected with dCas13a-MTD or the catalytic-null dCas13a-MTDD395A, with the nontargeting crNT as a control, or with the crRNA array from A. m6A level is normalized to the crNT transfection. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. C, as in B, but using dCas13a-FTO and the catalytic-null dCas13a-FTOY108A. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. D, as in B, individual vectors containing a single crRNA was used instead of the crRNA array. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. E, as in B, but using dCas13a-FTO and the catalytic-null dCas13a-FTOY108A, and individual vectors containing a single crRNA was used instead of the crRNA array and using dCas13a-FTO and the catalytic-null dCas13a-FTOY108A. Dots indicate the mean for each biological replicate, and the bar is the mean of all biological replicates, n = 3 biological replicates with three technical replicates each. crRNA, CRISPR RNA; m6A, N6-methyladenosine; MTD, methyltransferase domain.

      Discussion

      In this study, we have designed a CRISPR/dCas13a-associated system to edit the m6A methylation of circular RNA, mRNA, and lncRNAs. The catalytic-dead LwaCas13a (dCas13a) was fused to either the full-length m6A methyltransferase protein METTL3 (dCas13a-METTL3), the catalytical domain of METTL3 (dCas13a-MTD), or the demethylase protein FTO (dCas13a-FTO) to alter m6A modification status of targeted RNA sequences. We generated vectors suitable for liposomal or lentiviral transfection into cells. We validated the systems by exploring the impact of dCas13a fusions on m6A levels and the translation of GFP from an exogenous circular GFP reporter. The dCas13a fusions can also modulate m6A levels on linear endogenous transcripts, and these levels can impact transcript half-life. dCas13a is not perfect at targeting RNAs, and there is a possibility for off-target effects. We have demonstrated that the dCas13a fusions do not alter global levels of m6A, and overexpression did not affect endogenous levels of METTL3 or FTO. However, we cannot rule out other transcript-specific off-target effects. In the experiments reported here, the dCas13a fusions were expressed at around ∼20% of the endogenous levels of the corresponding proteins. It is possible the fusions work as dominant positives if expressed at higher levels or in cells with lower expression levels of the endogenous proteins.
      Several research groups have demonstrated a role for m6A in modulating transcript half-life; however, a direct relationship between half-life and m6A has been challenging to demonstrate because of the widespread marking of transcripts with m6A, and its role in many biological processes. Here, we utilized our m6A editors to specifically alter the level of m6A on several RNAs. Using the dCas13a fusions, we manipulated the levels of m6A on specific transcripts, and this led to a change in transcript half-life. These experiments help establish a direct relationship between m6A levels and transcript half-life.
      We also altered X chromosome activation status by editing m6A on XIST. This is a key result that demonstrates that m6A on XIST is crucial in enabling X chromosome suppression. Intriguingly, dCas13a-MTD could induce good suppression of X chromosome genes, suggesting m6A on XIST is required for initiation of X chromosome silencing. Knockdowns of METTL3 can potently reactivate X chromosome genes (
      • Patil D.P.
      • Chen C.K.
      • Pickering B.F.
      • Chow A.
      • Jackson C.
      • Guttman M.
      • et al.
      m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
      ); however, our dCas13a-FTO/crXIST experiment led only to a modest, but significant, reactivation. Potentially, the loss of m6A on XIST has only a weak ability to overcome other X chromosome epigenetic suppression mechanisms. In addition, reductions in m6A on other transcripts may be required to achieve robust X chromosome reactivation. Previous studies exploring the role of m6A on XIST used whole-genome KOs or knockdowns of m6A-modulating enzymes, which will alter the m6A levels of several thousand transcripts that may indirectly modulate X chromosome inactivation. Here, we demonstrate that deposition of m6A on XIST alone is capable of silencing genes on the X chromosome, and removal of m6A on XIST can modestly reactivate X chromosome genes.
      One interesting observation relates to the range of the dCas13a fusions and how far from the crRNA-binding site they can edit m6A. In the circular RNA, the m6A close to the ATG was altered even if the crRNA site was hundreds of nucleotides away. Conversely, in the linear XIST RNA experiments, all three crXISTs led to X gene activation or repression as appropriate; however, only when the crRNA was close to the m6A site did m6A levels change. This effect was the same for both dCas13a-METTL3 and dCas13a-FTO. This suggests that the dCas13a fusions have a limited range in linear RNAs but a longer range in circular RNAs. Potentially, this is due to the three-dimensional folding of the RNAs, and it will be interesting to see if this is a generalizable observation.
      It is increasingly clear there is an intimate interdependence between epigenetic control of DNA and epitranscriptomic control of RNA. For example, m6A marks the majority of DNA:DNA:RNA R-loops in stem cells (
      • Abakir A.
      • Giles T.C.
      • Cristini A.
      • Foster J.M.
      • Dai N.
      • Starczak M.
      • et al.
      N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.
      ), and m6A has been identified as a critical regulator of chromatin structure, transregulation of gene expression, and stem cell differentiation (
      • Xu W.
      • Li J.
      • He C.
      • Wen J.
      • Ma H.
      • Rong B.
      • et al.
      METTL3 regulates heterochromatin in mouse embryonic stem cells.
      ,
      • Liu J.
      • Gao M.
      • He J.
      • Wu K.
      • Lin S.
      • Jin L.
      • et al.
      The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.
      ,
      • Li L.
      • Zhu S.
      • Shu W.
      • Guo Y.
      • Guan Y.
      • Zeng J.
      • et al.
      Characterization of metabolic patterns in mouse Oocytes during meiotic maturation.
      ). The dCas13a-fusion tools described here will be instrumental in dissecting these roles on specific individual transcripts and groups of transcripts. Utilizing this system, researchers can isolate the effects of m6A on individual transcripts to explore pluripotency, development, oncogenesis, and human disease.

      Experimental procedures

      dCas13a-METTL3, MTD, FTO plasmid construction

      dCas13a fused with METTL3, MTD, and FTO separately, two nuclear localization signals, one hemagglutinin tag, and P2A mCherry are added inside each vector (Fig. S1A). A full list of Cas13 crRNAs used in this work is given in Table S1. Note that the D395A catalytic-null mutant on MTD is numbered according to the full-length METTL3. The plasmid backbones used for the lipofectamine-compatible system were pST1374 (Addgene: #13426) and pC0040-LwaCas13a crRNA backbone (Addgene: #103851). For the lentivirus system, dCas13a-M/F&crRNA vector, pFUGW/lentiCas9 (Addgene: #63592) was used. For the multiple crRNA vector, pC0040-LwaCas13a crRNA backbone (Addgene: #103851) was used.

      Cell culture and transfection

      Cells were maintained at 37 °C with 5% CO2 in a humidified incubator and passaged every 2 to 3 days. WT HEK293T cells were cultured in high-glucose Dulbecco's modified Eagle's medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (Gibco). Cells were split with TrypLE Express (Life Technologies) according to the manufacturer’s instructions. HEK293T cells were seeded on 12-well poly (d-lysine) plates (Corning) in culture medium. At 80% confluency, approximately 12 h after plating, cells were transfected with 1250 ng of dCas13a m6A editor plasmid and 1250 ng of crRNA plasmid using 5 μl of Lipofectamine 3000 (Thermo Fisher Scientific) in Opti-MEM I Reduced Serum Media (Thermo Fisher Scientific). Mettl3 KO mESCs were a kind gift from Jacob Hanna’s laboratory. The KO mESCs and matching WT mESC lines were cultured in fetal bovine serum–free N2B27-based media (
      • Gafni O.
      • Weinberger L.
      • Mansour A.A.
      • Manor Y.S.
      • Chomsky E.
      • Ben-Yosef D.
      • et al.
      Derivation of novel human ground state naive pluripotent stem cells.
      ). About 500 ml of N2B27 media was generated by including 240 ml Dulbecco's modified Eagle's medium/F12 (Biological Industries—Hepes free, custom made), 240 ml neurobasal (Invitrogen; catalog no.: 21103), 5 ml N2 supplement (Invitrogen; catalog no.: 17502048 or in house prepared), 5 ml B27 supplement (Invitrogen; catalog no.: 17504044), 1 mM glutamine (Invitrogen), 1% nonessential amino acids (Invitrogen), 0.1 mM β-mercaptoethanol (Sigma), and penicillin–streptomycin (Invitrogen). Naïve conditions for mESC included 10 μg recombinant human LIF (Peprotech) and small-molecule inhibitors CHIR99021 (CH, 1–3 μM; Axon Medchem) and PD0325901 (PD, 1 μM; TOCRIS) termed 2i. mESCs were cultured on fibronectin-coated plates where indicated. Naïve pluripotent cultures were passaged following 0.25% trypsinization every 3 to 4 days.

      Lentivirus production

      To produce lentivirus, HEK293T cells were transiently transfected with lentivirus constructs and cotransfected with the packaging plasmids pCMV-dR8.91 and pMD2.G. Lentivirus was collected 48 h after transfection and filtered through 0.45 μm filters. The virus supernatant was centrifuged at 1500g for 30 min at 4 °C to collect pellets. The pellets were resuspended in cold culture medium and directly added to cells or frozen at −80 °C. The resulting lentiviral particles were used to generate mESCs stably expressing Cas13a m6A editors. The mESCs were transduced with lentivirus pU6-crRNA-EF1a-dCas13a-METL3/FTO-2A-mCherry.

      RNA isolation and RT–qPCR

      Total RNA was isolated from WT or transiently transfected cells with MiniBEST Universal RNA Extraction Kit (Takara), an additional DNase I (NEB) digestion step was performed on all samples to avoid DNA contamination, and RNA concentration was measured by Nanodrop (Thermo Fisher Scientific). Total RNA was extracted using MiniBEST Universal RNA Extraction Kit. First-strand cDNA was synthesized by reverse transcription of 1 μg RNA using PrimeScript RT Master Mix (Takara). Quantitative real-time PCR was performed using TB Green Premix Ex Taq (Takara) in QuantStudio 7 Flex Real-Time PCR System (Life Technologies). βACTIN and GAPDH were used as reference genes for input normalization. The mRNA expression was measured by qPCR using the ΔΔCT method. Primers for qPCR are listed in Table S1.

      METTL3 knockdown via shRNA

      The shRNA targeting METTL3 used in this study was previously described (
      • Wang X.
      • Lu Z.
      • Gomez A.
      • Hon G.C.
      • Yue Y.
      • Han D.
      • et al.
      N6-methyladenosine-dependent regulation of messenger RNA stability.
      ,
      • Lin S.
      • Choe J.
      • Du P.
      • Triboulet R.
      • Gregory R.I.
      The m(6)A methyltransferase METTL3 promotes translation in human cancer cells.
      ) and was cloned into an shRNA expression backbone vector pSH. At 80% confluency, approximately 12 h after plating, cells were transfected with 2500 ng of shMETTL3 plasmid and 2500 ng of shControl (luciferase) plasmid separately with 5 μl of Lipofectamine 3000 in Opti-MEM I Reduced Serum Media. Cells were maintained at 70 to 80% confluency and collected 48 h after the transfection. Knockdown was confirmed by RT–qPCR and Western blot.

      RNA half-life assay

      HEK293T cells and mESCs for lifetime assay were cultured in 12-well plates, cultured cells were transfected with the dCas13a fusions including different crRNAs, and the control crRNA separately at 50% confluency. After 12 h, each well of a 12-well plate was reseeded into three wells in a 12-well plate, and each well was controlled to contain the same number of cells. After 48 h, actinomycin D was added at a concentration of 5 μg/ml at 4, 2, and 0 h before total RNA was extracted by MiniBEST Universal RNA Extraction Kit. The abundances of the target genes were measured at each time point by RT–qPCR using GAPDH as a reference gene. The degradation rate of RNA, k, was calculated by:
      log2(AtA0)=kt


      where t is transcription inhibition time (h), At and A0 represent mRNA quantity (attomole) at time t and time 0. Two k values were calculated: time 2 h versus time 0 h and time 4 h versus time 0 h. The final half-life was calculated by using the average of k 2 h and k 4 h:
      t12=kt2h+kt4h2


      SELECT assay

      The SELECT assay was performed as previously described (
      • Xiao Y.
      • Wang Y.
      • Tang Q.
      • Wei L.
      • Zhang X.
      • Jia G.
      An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6) -methyladenosine modification.
      ). Briefly, 80 fmol synthesized RNA oligo was mixed with 40 nM Up-T Primer and 40 nM Down Primer in 18 μl 1× reaction buffer. 1× CutSmart buffer (50 mM KAc (Ac=CH3COO), 20 mM Tris–HAc, 10 mM MgAc2, 100 μg/ml bovine serum albumin, pH 7.9 @ 25 °C) was used to examine SplintR ligase, T4 DNA ligase, and T4 RNA ligase 2 (dsRNA ligase). 1× T3 DNA ligase reaction buffer (66 mM Tris–HCl, 10 mM MgCl2, 1 mM ATP, 1 mM DTT, 7.5% PEG 6000, pH 7.6 @ 25 °C) was used with T3 DNA ligase and T7 DNA ligase. 1× 9°N DNA ligase reaction buffer (10 mM Tris–HCl, 600 μM ATP, 2.5 mM MgCl2, 2.5 mM DTT, 0.1% Triton X-100, pH 7.5 @ 25 °C) was used. 1× Taq DNA ligase reaction buffer (20 mM Tris–HCl, 25 mM KAc, 10 mM MgAc2, 10 mM DTT, 1 mM NAD, 0.1% Triton X-100, pH 7.6 @ 25 °C) was used in the test of Taq DNA ligase. The RNA and primers were annealed by incubating the mixture at a temperature gradient: 90 °C for 1 min, 80 °C for 1 min, 70 °C for 1 min, 60 °C for 1 min, 50 °C for 1 min, and then 40 °C for 6 min. A 2 μl mixture containing ligase with indicated concentration and 10 nmol ATP (only added in the test of SplintR ligase, T4 DNA ligase, and T4 RNA ligase 2) was added to the former annealed mixture. The final reaction mixture was incubated at 37 °C for 20 min and then denatured at 95 °C for 5 min and kept at 4 °C. Subsequently, RT–qPCR was carried out as described previously. SELECT primers are listed in Table S1.

      Western blot

      Whole-cell extracts were extracted by directly lysing the cells with 1× radioimmunoprecipitation assay buffer (Beyotime) with 1 mM PMSF (Beyotime) added immediately before use. Samples were boiled by adding 6× SDS sample buffer for 10 min at 100 °C and resolved using SDS-polyacrylamide gel electrophoresis. The proteins were probed with the following antibodies: monoclonal anti-GFP (1:2000 dilution; Thermo Fisher Scientific), anti-βACTIN (1:2000 dilution; Thermo Fisher Scientific), and anti-METTL3 (1:1000 dilution; Abcam), and anti-FTO (1:2000 dilution; Abcam). Immunodetection was performed using horseradish peroxidase–conjugated Affinipure goat anti-mouse IgG (H + L) (1:5000 dilution; catalog no.: SA0 001-1; Proteintech) or horseradish peroxidase–conjugated Affinipure goat anti-Rabbit IgG (H + L) (1:5000 dilution; catalog no.: SA00001-2; Proteintech) and ECL prime substrate (Bio-Rad) according to the manufacturer's instructions.

      Whole transcriptome m6A measurements

      Global m6A/m in total RNA was quantified by the EpiQuik m6A/m RNA Methylation Quantification Kit (Epigentek Group) following the manufacturers’ specifications and using 100 ng as input.

      m6A MeRIP–qPCR

      MeRIP–qPCR was performed using an EpiMark N6-Methyladenosine Enrichment Kit (catalog no. E1610S; New England Biolabs), according to the manufacturer’s instructions with some modifications. Briefly, total RNA was fragmented in a solution of 50 mM Tris–HCl, pH 8.0, 50 mM MgCl2, and heated at 95 °C for 8 min. The m6A-modified and m6A-unmodified control RNAs were spiked into the fragmented RNA, and a portion was saved as input RNA. The remaining fragmented RNA was subjected to m6A IP: 30 μl of protein G magnetic beads were washed twice by IP reaction buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.5, 0.1% NP-40 in nuclease-free water), resuspended in 250 μl of reaction buffer, and tumbled with 5 μg of anti-m6A antibody at 4 °C overnight. After two washes in reaction buffer, the antibody–bead mixture was resuspended in 500 μl of the reaction mixture containing 10 μg of fragmented total RNA, 100 μl of reaction buffer, and 5 μl of RNasin Plus RNase Inhibitor (Promega), and incubated for at least 4 h at 4 °C. To remove unbound RNA, samples were washed 5× with each of the following buffers: reaction buffer (150 mM NaCl, 10 mM Tris–HCl, pH 7.5, 0.1% NP-40 in nuclease-free water), low-salt reaction buffer (50 mM NaCl, 10 mM Tris–HCl, pH 7.5, 0.1% NP-40 in nuclease-free water), and high-salt reaction buffer (500 mM NaCl, 10 mM Tris–HCl, pH 7.5, 0.1% NP-40 in nuclease-free water). RNA was eluted in RLT buffer (Qiagen) and purified with RNA Clean & Concentrator-5 kits (Zymo Research). Purified RNA was reverse transcribed with High-Capacity RNA-to-cDNA (Thermo Fisher Scientific) according to the manufacturer’s protocol. The resulting cDNA was preamplified with SsoAdvanced PreAmp Supermix (Bio-Rad) according to the manufacturer’s protocol. qPCR was performed with IQ Multiplex Powermix (Bio-Rad). All reactions were performed and quantified on a CFX96 Real-Time PCR Detection System (Bio-Rad). MeRIP–qPCR primers are listed in Table S1.

      Fluorescent-activated cell sorting–based analysis of cell cycle and cell death

      Cell cycle and cell death were measured using Cell Cycle and Apoptosis Detection Kit (Beyotime; catalog no.: 1052), according to the manufacturer’s instructions. Briefly, transfected 293T cells were grown on a well in a 6-well plate for 36 h. Cells were washed with cold PBS, fixed in 70% ethanol, and stored at 4 °C for subsequent cell cycle analysis. Fixed cells were washed with PBS once and then resuspended in 1 ml of propidium iodide staining reagent. Samples were incubated in the dark for 30 min before cell cycle analysis. The distribution of cells in the cell cycle was measured by flow cytometer (BD FACSCalibur), and quantitation of cell cycle distribution was performed using Multi-cycle Software (ModFit software). The percentage of cells in the G1, S, and G2 phases and cell death was calculated.

      Statistical procedures

      Significance was calculated from a two-tailed unpaired Student’s t test for all indicated figure panels with a p value except for Figures 1, F and G and S5, E and F. Significance tests were calculated using GraphPad Prism 8 (GraphPad Software, Inc). Biological replicates were defined as experiments performed using different cells on different days. Technical replicates were defined as multiple repeats of the same biological replicate in PCR-based assays (SELECT, MeRIP–qPCR, and RT–qPCR).

      Data availability

      All data are contained within the article. Plasmids will be available upon request.

      Supporting information

      This article contains supporting information (
      • Meyer K.D.
      • Saletore Y.
      • Zumbo P.
      • Elemento O.
      • Mason C.E.
      • Jaffrey S.R.
      Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
      ,
      • Wang Y.
      • Xiao Y.
      • Dong S.
      • Yu Q.
      • Jia G.
      Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
      ).

      Conflict of interest

      The authors declare that they have no conflicts of interest with the contents of this article.

      Acknowledgments

      We thank Jacob Hanna for the kind gift of the Mettl3 KO mESCs. This work was supported by the National Natural Science Foundation of China (grant nos.: 31970589 and 32150710521 ), the Shenzhen Innovation Committee of Science and Technology (grant no.: ZDSYS20200811144002008 ), the Stable Support Plan Program of Shenzhen Natural Science Fund (grant no.: 20200925153035002 ), the University of Macau (grant no.: MYRG2020-00100-FHS ), and the Macau Science and Technology Development Fund (grant no.: 0011/2019/AKP ). This work was supported by the Center for Computational Science and Engineering of Southern University of Science and Technology .

      Author contributions

      C. C. and A. H. conceptualization; G. M. methodology; C. C. and A. H. formal analysis; C. C. investigation; C. C. writing–original draft; E. C. and A. H. writing–review & editing; E. C. and A. H. supervision; E. C. and A. H. project administration; E. C. and A. H. funding acquisition.

      References

        • Fu Y.
        • Dominissini D.
        • Rechavi G.
        • He C.
        Gene expression regulation mediated through reversible m6A RNA methylation.
        Nat. Rev. Genet. 2014; 15: 293-306
        • Roignant J.Y.
        • Soller M.
        m(6)A in mRNA: an ancient mechanism for fine-tuning gene expression.
        Trends Genet. 2017; 33: 380-390
        • Desrosiers R.
        • Friderici K.
        • Rottman F.
        Identification of methylated nucleosides in messenger RNA from novikoff hepatoma cells.
        Proc. Natl. Acad. Sci. U. S. A. 1974; 71: 3971-3975
        • Meyer K.D.
        • Saletore Y.
        • Zumbo P.
        • Elemento O.
        • Mason C.E.
        • Jaffrey S.R.
        Comprehensive analysis of mRNA methylation reveals enrichment in 3' UTRs and near stop codons.
        Cell. 2012; 149: 1635-1646
        • Dominissini D.
        • Moshitch-Moshkovitz S.
        • Schwartz S.
        • Salmon-Divon M.
        • Ungar L.
        • Osenberg S.
        • et al.
        Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.
        Nature. 2012; 485: 201-206
        • Coker H.
        • Wei G.
        • Brockdorff N.
        m6A modification of non-coding RNA and the control of mammalian gene expression.
        Biochim. Biophys. Acta Gene Regul. Mech. 2019; 1862: 310-318
        • Patil D.P.
        • Chen C.K.
        • Pickering B.F.
        • Chow A.
        • Jackson C.
        • Guttman M.
        • et al.
        m(6)A RNA methylation promotes XIST-mediated transcriptional repression.
        Nature. 2016; 537: 369-373
        • Petropoulos S.
        • Edsgard D.
        • Reinius B.
        • Deng Q.
        • Panula S.P.
        • Codeluppi S.
        • et al.
        Single-cell RNA-seq reveals lineage and X chromosome dynamics in human preimplantation embryos.
        Cell. 2016; 165: 1012-1026
        • Wang X.
        • Lu Z.
        • Gomez A.
        • Hon G.C.
        • Yue Y.
        • Han D.
        • et al.
        N6-methyladenosine-dependent regulation of messenger RNA stability.
        Nature. 2014; 505: 117-120
        • Liu J.
        • Dou X.
        • Chen C.
        • Chen C.
        • Liu C.
        • Xu M.M.
        • et al.
        N6-methyladenosine of chromosome-associated regulatory RNA regulates chromatin state and transcription.
        Science. 2020; 367: 580-586
        • Mauer J.
        • Luo X.
        • Blanjoie A.
        • Jiao X.
        • Grozhik A.V.
        • Patil D.P.
        • et al.
        Reversible methylation of m(6)Am in the 5' cap controls mRNA stability.
        Nature. 2017; 541: 371-375
        • Geula S.
        • Moshitch-Moshkovitz S.
        • Dominissini D.
        • Mansour A.A.
        • Kol N.
        • Salmon-Divon M.
        • et al.
        Stem cells. m6A mRNA methylation facilitates resolution of naive pluripotency toward differentiation.
        Science. 2015; 347: 1002-1006
        • Yu J.
        • Chen M.
        • Huang H.
        • Zhu J.
        • Song H.
        • Zhu J.
        • et al.
        Dynamic m6A modification regulates local translation of mRNA in axons.
        Nucl. Acids Res. 2018; 46: 1412-1423
        • Xiang J.F.
        • Yang Q.
        • Liu C.X.
        • Wu M.
        • Chen L.L.
        • Yang L.
        N(6)-Methyladenosines modulate A-to-I RNA editing.
        Mol. Cell. 2018; 69: 126-135.e126
        • Barbieri I.
        • Tzelepis K.
        • Pandolfini L.
        • Shi J.
        • Millan-Zambrano G.
        • Robson S.C.
        • et al.
        Promoter-bound METTL3 maintains myeloid leukaemia by m(6)A-dependent translation control.
        Nature. 2017; 552: 126-131
        • Shi H.
        • Zhang X.
        • Weng Y.L.
        • Lu Z.
        • Liu Y.
        • Lu Z.
        • et al.
        m(6)A facilitates hippocampus-dependent learning and memory through YTHDF1.
        Nature. 2018; 563: 249-253
        • Xiao W.
        • Adhikari S.
        • Dahal U.
        • Chen Y.S.
        • Hao Y.J.
        • Sun B.F.
        • et al.
        Nuclear m(6)A reader YTHDC1 regulates mRNA splicing.
        Mol. Cell. 2016; 61: 507-519
        • Wei J.
        • He C.
        Chromatin and transcriptional regulation by reversible RNA methylation.
        Curr. Opin. Cell Biol. 2021; 70: 109-115
        • Batista P.J.
        • Molinie B.
        • Wang J.
        • Qu K.
        • Zhang J.
        • Li L.
        • et al.
        m(6)A RNA modification controls cell fate transition in mammalian embryonic stem cells.
        Cell Stem Cell. 2014; 15: 707-719
        • Lin S.
        • Choe J.
        • Du P.
        • Triboulet R.
        • Gregory R.I.
        The m(6)A methyltransferase METTL3 promotes translation in human cancer cells.
        Mol. Cell. 2016; 62: 335-345
        • Deng X.
        • Su R.
        • Weng H.
        • Huang H.
        • Li Z.
        • Chen J.
        RNA N(6)-methyladenosine modification in cancers: current status and perspectives.
        Cell Res. 2018; 28: 507-517
        • Liu J.
        • Eckert M.A.
        • Harada B.T.
        • Liu S.M.
        • Lu Z.
        • Yu K.
        • et al.
        m(6)A mRNA methylation regulates AKT activity to promote the proliferation and tumorigenicity of endometrial cancer.
        Nat. Cell Biol. 2018; 20: 1074-1083
        • Li Z.
        • Weng H.
        • Su R.
        • Weng X.
        • Zuo Z.
        • Li C.
        • et al.
        FTO plays an oncogenic role in acute myeloid leukemia as a N(6)-methyladenosine RNA demethylase.
        Cancer Cell. 2017; 31: 127-141
        • Li Z.
        • Peng Y.
        • Li J.
        • Chen Z.
        • Chen F.
        • Tu J.
        • et al.
        N6-methyladenosine regulates glycolysis of cancer cells through PDK4.
        Nat. Commun. 2020; 11: 2578
        • Engel M.
        • Eggert C.
        • Kaplick P.M.
        • Eder M.
        • Roh S.
        • Tietze L.
        • et al.
        The role of m(6)A/m-RNA methylation in stress response regulation.
        Neuron. 2018; 99: 389-403.e389
        • Zhang Z.
        • Wang M.
        • Xie D.
        • Huang Z.
        • Zhang L.
        • Yang Y.
        • et al.
        METTL3-mediated N(6)-methyladenosine mRNA modification enhances long-term memory consolidation.
        Cell Res. 2018; 28: 1050-1061
        • Chu C.
        • Zhang Q.C.
        • da Rocha S.T.
        • Flynn R.A.
        • Bharadwaj M.
        • Calabrese J.M.
        • et al.
        Systematic discovery of Xist RNA binding proteins.
        Cell. 2015; 161: 404-416
        • Huisman B.
        • Manske G.
        • Carney S.
        • Kalantry S.
        Functional dissection of the m6A RNA modification.
        Trends Biochem. Sci. 2017; 42: 85-86
        • Brown C.J.
        • Hendrich B.D.
        • Rupert J.L.
        • Lafrenière R.G.
        • Xing Y.
        • Lawrence J.
        • et al.
        The human XIST gene: analysis of a 17 kb inactive X-specific RNA that contains conserved repeats and is highly localized within the nucleus.
        Cell. 1992; 71: 527-542
        • Engreitz J.M.
        • Ollikainen N.
        • Guttman M.
        Long non-coding RNAs: spatial amplifiers that control nuclear structure and gene expression.
        Nat. Rev. Mol. Cell Biol. 2016; 17: 756-770
        • Roundtree I.A.
        • Evans M.E.
        • Pan T.
        • He C.
        Dynamic RNA modifications in gene expression regulation.
        Cell. 2017; 169: 1187-1200
        • Yang Y.
        • Hsu P.J.
        • Chen Y.S.
        • Yang Y.G.
        Dynamic transcriptomic m(6)A decoration: Writers, erasers, readers and functions in RNA metabolism.
        Cell Res. 2018; 28: 616-624
        • Zaccara S.
        • Jaffrey S.R.
        A unified model for the function of YTHDF proteins in regulating m6A-modified mRNA.
        Cell. 2020; 181: 1582-1595.e1518
        • Terns M.P.
        • Terns R.M.
        CRISPR-based adaptive immune systems.
        Curr. Opin. Microbiol. 2011; 14: 321-327
        • Bhaya D.
        • Davison M.
        • Barrangou R.
        CRISPR-cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation.
        Annu. Rev. Genet. 2011; 45: 273-297
        • Barrangou R.
        • Fremaux C.
        • Deveau H.
        • Richards M.
        • Boyaval P.
        • Moineau S.
        • et al.
        CRISPR provides acquired resistance against viruses in prokaryotes.
        Science. 2007; 315: 1709-1712
        • Koonin E.V.
        • Makarova K.S.
        • Zhang F.
        Diversity, classification and evolution of CRISPR-Cas systems.
        Curr. Opin. Microbiol. 2017; 37: 67-78
        • Knott G.J.
        • Doudna J.A.
        CRISPR-Cas guides the future of genetic engineering.
        Science. 2018; 361: 866-869
        • Komor A.C.
        • Badran A.H.
        • Liu D.R.
        CRISPR-based Technologies for the manipulation of eukaryotic genomes.
        Cell. 2017; 168: 20-36
        • Wright A.V.
        • Nunez J.K.
        • Doudna J.A.
        Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering.
        Cell. 2016; 164: 29-44
        • Goell J.H.
        • Hilton I.B.
        CRISPR/Cas-Based epigenome editing: advances, applications, and clinical utility.
        Trends Biotechnol. 2021; 39: 678-691
        • Jinek M.
        • Chylinski K.
        • Fonfara I.
        • Hauer M.
        • Doudna J.A.
        • Charpentier E.
        A programmable dual-RNA guided DNA endonuclease in adaptive bacterial immunity.
        Science. 2012; 337: 816-821
        • Cong L.
        • Ran F.A.
        • Cox D.
        • Lin S.
        • Barretto R.
        • Habib N.
        • et al.
        Multiplex genome engineering using CRISPR/Cas systems.
        Science. 2013; 339: 819-823
        • Mali P.
        • Yang L.
        • Esvelt K.M.
        • Aach J.
        • Guell M.
        • DiCarlo J.E.
        • et al.
        RNA-guided human genome engineering via Cas9.
        Science. 2013; 339: 823-826
        • Makarova K.S.
        • Zhang F.
        • Koonin E.V.
        SnapShot: class 1 CRISPR-cas systems.
        Cell. 2017; 168: 946-946.e941
        • Makarova K.S.
        • Zhang F.
        • Koonin E.V.
        SnapShot: class 2 CRISPR-cas systems.
        Cell. 2017; 168: 328-328.e321
        • Abudayyeh O.O.
        • Gootenberg J.S.
        • Essletzbichler P.
        • Han S.
        • Joung J.
        • Belanto J.J.
        • et al.
        RNA targeting with CRISPR–Cas13.
        Nature. 2017; 550: 280-284
        • East-Seletsky A.
        • O'Connell M.R.
        • Knight S.C.
        • Burstein D.
        • Cate J.H.
        • Tjian R.
        • et al.
        Two distinct RNase activities of CRISPR-C2c2 enable guide-RNA processing and RNA detection.
        Nature. 2016; 538: 270-273
        • Liu L.
        • Li X.
        • Wang J.
        • Wang M.
        • Chen P.
        • Yin M.
        • et al.
        Two distant catalytic sites are responsible for C2c2 RNase activities.
        Cell. 2017; 168: 121-134.e112
        • Abudayyeh O.O.
        • Gootenberg J.S.
        • Konermann S.
        • Joung J.
        • Slaymaker I.M.
        • Cox D.B.
        • et al.
        C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector.
        Science. 2016; 353: aaf5573
        • Konermann S.
        • Lotfy P.
        • Brideau N.J.
        • Oki J.
        • Shokhirev M.N.
        • Hsu P.D.
        Transcriptome engineering with RNA-targeting type VI-D CRISPR effectors.
        Cell. 2018; 173: 665-676.e614
        • Smargon A.A.
        • Shi Y.J.
        • Yeo G.W.
        RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering.
        Nat. Cell Biol. 2020; 22: 143-150
        • Cox D.B.T.
        • Gootenberg J.S.
        • Abudayyeh O.O.
        • Franklin B.
        • Kellner M.J.
        • Joung J.
        • et al.
        RNA editing with CRISPR-Cas13.
        Science. 2017; 358: 1019-1027
        • Wilson C.
        • Chen P.J.
        • Miao Z.
        • Liu D.R.
        Programmable m6A modification of cellular RNAs with a Cas13-directed methyltransferase.
        Nat. Biotechnol. 2020; 38: 1431-1440
        • Liu X.M.
        • Zhou J.
        • Mao Y.
        • Ji Q.
        • Qian S.B.
        Programmable RNA N(6)-methyladenosine editing by CRISPR-Cas9 conjugates.
        Nat. Chem. Biol. 2019; 15: 865-871
        • Li J.
        • Chen Z.
        • Chen F.
        • Xie G.
        • Ling Y.
        • Peng Y.
        • et al.
        Targeted mRNA demethylation using an engineered dCas13b-ALKBH5 fusion protein.
        Nucl. Acids Res. 2020; 48: 5684-5694
        • Chen X.
        • Zhao Q.
        • Zhao Y.-L.
        • Chai G.-S.
        • Cheng W.
        • Zhao Z.
        • et al.
        Targeted RNA N6-methyladenosine demethylation controls cell fate transition in human pluripotent stem cells.
        Adv. Sci. 2021; 8e2003902
        • Mo J.
        • Chen Z.
        • Qin S.
        • Li S.
        • Liu C.
        • Zhang L.
        • et al.
        Trades: targeted RNA demethylation by SunTag system.
        Adv. Sci. (Weinh). 2020; 72001402
        • Xia Z.
        • Tang M.
        • Ma J.
        • Zhang H.
        • Gimple R.C.
        • Prager B.C.
        • et al.
        Epitranscriptomic editing of the RNA N6-methyladenosine modification by dCasRx conjugated methyltransferase and demethylase.
        Nucl. Acids Res. 2021; 49: 7361-7374
        • Zhao J.
        • Li B.
        • Ma J.
        • Jin W.
        • Ma X.
        Photoactivatable RNA N(6) -methyladenosine editing with CRISPR-cas13.
        Small. 2020; 16e1907301
        • Sun X.
        • Wang D.O.
        • Wang J.
        Targeted manipulation of m(6)A RNA modification through CRISPR-Cas-based strategies.
        Methods. 2022; 203: 56-61
        • Huang J.
        • Dong X.
        • Gong Z.
        • Qin L.Y.
        • Yang S.
        • Zhu Y.L.
        • et al.
        Solution structure of the RNA recognition domain of METTL3-METTL14 N(6)-methyladenosine methyltransferase.
        Protein Cell. 2019; 10: 272-284
        • Wang X.
        • Feng J.
        • Xue Y.
        • Guan Z.
        • Zhang D.
        • Liu Z.
        • et al.
        Structural basis of N(6)-adenosine methylation by the METTL3-METTL14 complex.
        Nature. 2016; 534: 575-578
        • Śledź P.
        • Jinek M.
        Structural insights into the molecular mechanism of the m(6)A writer complex.
        Elife. 2016; 5e18434
        • Wang P.
        • Doxtader Katelyn A.
        • Nam Y.
        Structural basis for cooperative function of Mettl3 and Mettl14 methyltransferases.
        Mol. Cell. 2016; 63: 306-317
        • Fu Y.
        • Jia G.
        • Pang X.
        • Wang R.N.
        • Wang X.
        • Li C.J.
        • et al.
        FTO-mediated formation of N6-hydroxymethyladenosine and N6-formyladenosine in mammalian RNA.
        Nat. Commun. 2013; 4: 1798
        • Han Z.
        • Niu T.
        • Chang J.
        • Lei X.
        • Zhao M.
        • Wang Q.
        • et al.
        Crystal structure of the FTO protein reveals basis for its substrate specificity.
        Nature. 2010; 464: 1205-1209
        • Zhang X.
        • Wei L.H.
        • Wang Y.
        • Xiao Y.
        • Liu J.
        • Zhang W.
        • et al.
        Structural insights into FTO's catalytic mechanism for the demethylation of multiple RNA substrates.
        Proc. Natl. Acad. Sci. U. S. A. 2019; 116: 2919-2924
        • Zheng G.
        • Dahl J.A.
        • Niu Y.
        • Fedorcsak P.
        • Huang C.M.
        • Li C.J.
        • et al.
        ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility.
        Mol. Cell. 2013; 49: 18-29
        • Xu C.
        • Liu K.
        • Tempel W.
        • Demetriades M.
        • Aik W.
        • Schofield C.J.
        • et al.
        Structures of human ALKBH5 demethylase reveal a unique binding mode for specific single-stranded N6-methyladenosine RNA demethylation.
        J. Biol. Chem. 2014; 289: 17299-17311
        • Feng C.
        • Liu Y.
        • Wang G.
        • Deng Z.
        • Zhang Q.
        • Wu W.
        • et al.
        Crystal structures of the human RNA demethylase Alkbh5 reveal basis for substrate recognition.
        J. Biol. Chem. 2014; 289: 11571-11583
        • Liang D.
        • Wilusz J.E.
        Short intronic repeat sequences facilitate circular RNA production.
        Genes Dev. 2014; 28: 2233-2247
        • Wang Y.
        • Wang Z.
        Efficient backsplicing produces translatable circular mRNAs.
        RNA. 2015; 21: 172-179
        • Yang Y.
        • Fan X.
        • Mao M.
        • Song X.
        • Wu P.
        • Zhang Y.
        • et al.
        Extensive translation of circular RNAs driven by N(6)-methyladenosine.
        Cell Res. 2017; 27: 626-641
        • Linder B.
        • Grozhik A.V.
        • Olarerin-George A.O.
        • Meydan C.
        • Mason C.E.
        • Jaffrey S.R.
        Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.
        Nat. Met. 2015; 12: 767-772
        • Xiao Y.
        • Wang Y.
        • Tang Q.
        • Wei L.
        • Zhang X.
        • Jia G.
        An elongation- and ligation-based qPCR amplification method for the radiolabeling-free detection of locus-specific N(6) -methyladenosine modification.
        Angew. Chem. Int. Ed. Engl. 2018; 57: 15995-16000
        • Fu Y.
        • Zhuang X.
        m6A-binding YTHDF proteins promote stress granule formation.
        Nat. Chem. Biol. 2020; 16: 955-963
        • Ji P.
        • Wang X.
        • Xie N.
        • Li Y.
        N6-Methyladenosine in RNA and DNA: an epitranscriptomic and epigenetic player implicated in determination of stem cell fate.
        Stem Cells Int. 2018; 20183256524
        • Sun L.
        • Fu X.
        • Ma G.
        • Hutchins A.P.
        Chromatin and epigenetic rearrangements in embryonic stem cell fate transitions.
        Front. Cell Dev. Biol. 2021; 9: 637309
        • Panning B.
        • Dausman J.
        • Jaenisch R.
        X chromosome inactivation is mediated by Xist RNA stabilization.
        Cell. 1997; 90: 907-916
        • Penny G.D.
        • Kay G.F.
        • Sheardown S.A.
        • Rastan S.
        • Brockdorff N.
        Requirement for Xist in X chromosome inactivation.
        Nature. 1996; 379: 131-137
        • Sheardown S.A.
        • Duthie S.M.
        • Johnston C.M.
        • Newall A.E.T.
        • Formstone E.J.
        • Arkell R.M.
        • et al.
        Stabilization of Xist RNA mediates initiation of X chromosome inactivation.
        Cell. 1997; 91: 99-107
        • Hernández-Muñoz I.
        • Lund A.H.
        • van der Stoop P.
        • Boutsma E.
        • Muijrers I.
        • Verhoeven E.
        • et al.
        Stable X chromosome inactivation involves the PRC1 Polycomb complex and requires histone MACROH2A1 and the CULLIN3/SPOP ubiquitin E3 ligase.
        Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 7635-7640
        • Jones A.N.
        • Tikhaia E.
        • Mourão A.
        • Sattler M.
        Structural effects of m6A modification of the Xist A-repeat AUCG tetraloop and its recognition by YTHDC1.
        Nucl. Acids Res. 2022; 50: 2350-2362
        • Lin Y.-C.
        • Boone M.
        • Meuris L.
        • Lemmens I.
        • Van Roy N.
        • Soete A.
        • et al.
        Genome dynamics of the human embryonic kidney 293 lineage in response to cell biology manipulations.
        Nat. Commun. 2014; 5: 4767
        • Abakir A.
        • Giles T.C.
        • Cristini A.
        • Foster J.M.
        • Dai N.
        • Starczak M.
        • et al.
        N6-methyladenosine regulates the stability of RNA:DNA hybrids in human cells.
        Nat. Genet. 2020; 52: 48-55
        • Xu W.
        • Li J.
        • He C.
        • Wen J.
        • Ma H.
        • Rong B.
        • et al.
        METTL3 regulates heterochromatin in mouse embryonic stem cells.
        Nature. 2021; 591: 317-321
        • Liu J.
        • Gao M.
        • He J.
        • Wu K.
        • Lin S.
        • Jin L.
        • et al.
        The RNA m6A reader YTHDC1 silences retrotransposons and guards ES cell identity.
        Nature. 2021; 591: 322-326
        • Li L.
        • Zhu S.
        • Shu W.
        • Guo Y.
        • Guan Y.
        • Zeng J.
        • et al.
        Characterization of metabolic patterns in mouse Oocytes during meiotic maturation.
        Mol. Cell. 2020; 80: 525-540.e529
        • Gafni O.
        • Weinberger L.
        • Mansour A.A.
        • Manor Y.S.
        • Chomsky E.
        • Ben-Yosef D.
        • et al.
        Derivation of novel human ground state naive pluripotent stem cells.
        Nature. 2013; 504: 282-286
        • Wang Y.
        • Xiao Y.
        • Dong S.
        • Yu Q.
        • Jia G.
        Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine.
        Nat. Chem. Biol. 2020; 16: 896-903