Skip to main content
NCBI home page
Plant Biotechnology Journal logoLink to Plant Biotechnology Journal
. 2024 Feb 16;22(7):1867–1880. doi: 10.1111/pbi.14307

Programmable RNA N6‐methyladenosine editing with CRISPR/dCas13a in plants

Chuanlin Shi 1, † , Wenli Zou 1, † , Xiangpei Liu 1, † , Hong Zhang 1, † , Xiaofang Li 1,2, Guiling Fu 1,3, Qili Fei 1,✉, Qian Qian 1,4,5,✉, Lianguang Shang 1,5,✉
PMCID: PMC11182597  PMID: 38363049

Summary

N6‐methyladenonsine (m6A) is the most prevalent internal modification of messenger RNA (mRNA) and plays critical roles in mRNA processing and metabolism. However, perturbation of individual m6A modification to reveal its function and the phenotypic effects is still lacking in plants. Here, we describe the construction and characterization of programmable m6A editing tools by fusing the m6A writers, the core catalytic domain of the MTA and MTB complex, and the AlkB homologue 5 (ALKBH5) eraser, to catalytically dead Cas13a (dCas13a) to edit individual m6A sites on mRNAs. We demonstrated that our m6A editors could efficiently and specifically deposit and remove m6A modifications on specific RNA transcripts in both Nicotiana benthamiana and Arabidopsis thaliana. Moreover, we found that targeting SHORT‐ROOT (SHR) transcripts with a methylation editor could significantly increase its m6A levels with limited off‐target effects and promote its degradation. This leads to a boost in plant growth with enlarged leaves and roots, increased plant height, plant biomass, and total grain weight in Arabidopsis. Collectively, these findings suggest that our programmable m6A editing tools can be applied to study the functions of individual m6A modifications in plants, and may also have potential applications for future crop improvement.

Keywords: N6‐methyladenonsine, m6A editing, CRISPR/dCas13a, plant growth, Arabidopsis

Introduction

RNA is a key molecule that transfers genetic information from DNA to proteins in living organisms. In recent decades, more than 100 chemical modifications have been identified in cellular RNA, including N6‐methyladenonsine (m6A), N1‐methyladenosine (m1A), 5‐methylcytosine (m5C) and pseudouridine (Ψ) (Boccaletto et al., 2022; Zhao et al., 2017). N6‐methyladenosine (m6A) is the most abundant internal modification of mRNA and is essential for post‐transcriptional gene regulation. It also plays critical roles in mRNA processing and metabolism, including RNA splicing, export and translocation, translation, and mRNA stability (Kasowitz et al., 2018; Meyer et al., 2015; Roundtree et al., 2017; Wang et al., 2014, 2015; Xiao et al., 2016). Recently, m6A modification has also been found to co‐transcriptionally regulate histone modification and chromatin status (Huang et al., 2019; Li et al., 2020b; Liu et al., 2020; Xu et al., 2021).

m6A modification of mRNA is reversible and dictated by three types of proteins: writers, erasers and readers. The modification is installed at the conserved consensus sequence, RRACH (R = G or A; H: U > A > C), as an m6A motif. In mammals, this modification is deposited on transcripts by a methyltransferase complex named m6A ‘writers’, which is composed of the methyltransferase‐like 3 (METTL3), METTL14, Wilm's tumour1‐associating protein (WTAP), KIAA1429/VIRMA and HAKAI (Bokar et al., 1997; Liu et al., 2014; Ping et al., 2014; Schwartz et al., 2014; Yue et al., 2018). The m6A modification can be removed by demethylases named m6A ‘erasers’, such as ALKBH5 and fat‐mass and obesity‐associated protein (FTO) (Jia et al., 2011; Zheng et al., 2013) in mammals. The modification can be recognized by the m6A ‘readers’, which are YT521‐B homology (YTH) domain‐containing proteins.

Because of evolutionary conservation, there are homologous m6A writers, erasers and reader genes in plants. In Arabidopsis thaliana, the core members of the m6A methyltransferase complex are MTA (METTL3 homologue), MTB (METTL14 homologue), FIP37 (WTAP homologue), VIRILIZER (KIAA1429 homologue) and HAKAI (HAKAI human homologue) (Ruzicka et al., 2017; Shen et al., 2016; Zhong et al., 2008). The depleted or reduced expression of these plant m6A writers can result in a diverse array of developmentally defective phenotypes. For example, knockout of MTA, FIP37 and VIRILIZER results in a significant decrease in global m6A levels, resulting in aberrant vascular formation in the root, reduced apical dominance, enlargement of vegetative shoot apical meristem, abnormal leaf development and embryonic lethality (Bodi et al., 2012; Ruzicka et al., 2017; Shen et al., 2016; Zhong et al., 2008). The m6A demethylases in Arabidopsis are ALKBH10B and ALKBH9B (Duan et al., 2017; Martinez‐Perez et al., 2017). Loss‐of‐function of ALKBH10B delays flowering, represses vegetative growth and shows hypersensitivity to stress during seed germination (Duan et al., 2017; Tang et al., 2021). There are 13 YTH domain‐containing paralogs that mediate the recognition of m6A in Arabidopsis, including ECT1‐12 and CPSF30 (Yue et al., 2019). ECT2 is one of the best‐characterized m6A reader proteins, and its binding sites are significantly enriched in the 3′ UTRs of target transcripts. The protein is responsible for regulating 3′ UTR processing and facilitating mRNA stability. Disruption of ECT2 influences trichome branching (Scutenaire et al., 2018; Wei et al., 2018). Recent studies have found that CPSF30‐L, the longer isoform of CPSF30, regulates mRNA degradation and flowering time by recognizing m6A‐modified far‐upstream elements (FUE) to determine polyadenylation site selection in liquid‐like nuclear bodies (Hou et al., 2021; Song et al., 2021). Together, these findings highlight the important roles of m6A in RNA metabolism and development in plants. However, the mechanism by which m6A modification of a single gene of interest regulates transcript metabolism and how it can affect plant development remains unclear.

Previous studies of m6A in plants have mainly focused on the knockout of key m6A writers, erasers or reader genes combined with transcriptome‐wide MeRIP‐sequencing (MeRIP‐seq) to identify global changes in m6A levels in thousands of target transcripts. To dissect the m6A function of an individual target gene of interest, tools that can programmatically add and remove m6A modifications at the base resolution are urgently needed. In recent years, CRISPR/Cas9‐based technologies have been widely engineered by fusing catalytically dead Cas9 (dCas9) with different enzymes for precision genome and epigenome editing for various purposes (Komor et al., 2017; Liu et al., 2019). CRISPR‐Cas13 protein has been found to exclusively binds to and cleave single‐stranded RNA aided by a complementary guide RNA (Abudayyeh et al., 2016, 2017; East‐Seletsky et al., 2016; Kordys et al., 2022), making it suitable for engineering applications in RNA research. Recently, several studies have developed programmable RNA m6A methylation and demethylation editing based on the CRISPR/dCas9 and CRISPR/dCas13 systems in bacteria and mammalian cells (Chen et al., 2021; Komor et al., 2017; Li et al., 2020a; Liu et al., 2019; Wilson et al., 2020; Xia et al., 2021). However, there are currently no reports of the precise manipulation of individual m6A levels for phenotypic studies at the organism level.

In the present study, we constructed and characterized RNA methylation and demethylation editing tools based on the CRISPR/dCas13 system to deposit and remove m6A modification on specific transcripts, respectively, in plants. We fused the core catalytic domain of the MTA and MTB complex, the m6A writers in Arabidopsis, to catalytically dead Cas13 from Leptotrichia shahii (dLshCas13a) and combined it with a guide RNA (gRNA) to generate the m6A methylation tool. We employed the same strategy to generate the m6A demethylation tool for ALKBH5, a m6A eraser in mammals. These m6A editing tools were successfully demonstrated in both Nicotiana benthamiana and Arabidopsis. Moreover, we found that targeting the transcript of SHR with the m6A methylation tool could significantly induce m6A levels in SHR and enhance its mRNA degradation, leading to an increase in leaf size, plant height, grain weight and aerial biomass. These results suggest that our editing tools could be used to improve plant traits. Our study provides programmable m6A methylation and demethylation tools for plants and demonstrates their potential applications for future crop improvement.

Results

Design of the programmable RNA methylation and demethylation editing tools

The CRISPR/LshCas13a system (formerly named C2c2) from Leptotrichia shahii was first used to bind and cleave RNA (Abudayyeh et al., 2016). It was soon engineered for use in the creation of virus‐resistant plants (Aman et al., 2018; Zhan et al., 2019; Zhang et al., 2019), indicating its promising application in plant science. To provide a simple tool for programmable m6A modification based on the CRISPR/LshCas13a system, we first developed a catalytically dead Cas13a (dCas13a) by mutagenesis of R597A, H602A, R1278A, and H1283A in the conserved RNA‐cleaving HEPN domains (Figure S1). A previous study revealed that a complex of truncated METTL3 and METTL14 which retained the core domains still possessed methyltransferase activity in mammals (Wilson et al., 2020). Based on evolutionary conservation, we selected the homologous methyltransferase domain of MTA (residues 446–667), ΔMTA446–667, and the MTA‐interacting domain of MTB (residues 562–876), ΔMTB562–876, in Arabidopsis to weaken the non‐specific RNA‐binding affinity of full‐length MTA and MTB. We connected truncated ΔMTA446–667 and ΔMTB562–876 with a flexible (GGS)10 linker to generate a fused methyltransferase complex (Figure S1). Next, we fused this simplified complex to the C‐terminus of dCas13a with a longer flexible (SGGS)2‐XTEN‐(SGGS)2 linker (Schellenberger et al., 2009), a nuclear export signal (NES), and a 3xHA tag to generate the dCas13a‐NES‐(SGGS)2‐XTEN‐(SGGS)2‐ΔMTA446–667‐(GGS)10‐ΔMTB562–876‐3xHA fusion protein. This fusion was used as the programmable m6A methylation editor (Figures 1a,b and S1) and is referred to as dcM. We also developed a catalytically dead ΔMTA (ΔMTAD482A) by mutagenizing the methyltransferase activity centre, generating an inactive m6A methylation editor, referred to as dcdM, for use as a negative control (Figures 1a,b and S1). Concurrently, an AtU6 promoter‐driven guide RNA for dCas13a was cloned into the construction for ease of use. To evaluate whether these constructs could be normally expressed in plant cells, we transformed them into Nicotiana benthamiana leaf cells. Western blot analysis showed that both dcM and dcdM fusions and the positive control dCas13a were successfully expressed in plant cells (Figure 1c).

Figure 1.

Figure 1

Open in a new tab

Design of the programmable m6A editing tools in plants. (a) Proposed strategy for the m6A methylation tool. (b) Schematic representation of the dcM and dcdM constructions. (c) Western blot analysis of dCas13a, dcM and dcdM fusion proteins expressed in tobacco leaf cells with anti‐HA antibody. dCas13a, a 3xHA‐tag fused in the C‐terminus of dCas13a served as a positive control. The control lance indicated protein extract from the tobacco leaf without infection. Ponceau staining served as loading control. (d) Proposed strategy for the m6A demethylation tool. (e) Schematic representation of the dcA and dcdA constructions. (f) Western blot analysis of dCas13a, dcA and dcdA fusion proteins expressed in tobacco leaf cells with anti‐HA antibody. Ponceau staining served as loading control.

Next, we developed a programmable m6A demethylation editor based on dCas13a in plants. Since the Arabidopsis demethylases ALKBH9B and ALKBH10B exhibit lower demethylase activity than the human orthologous ALKBH5 (Duan et al., 2017), in lieu, we used the human ALKBH5 to develop the m6A demethylation editor (Figure 1d). Employing the same strategy used to create dcM, we fused ALKBH5 to the C‐terminus of dCas13a to generate the dCas13a‐NES‐(SGGS)2‐XTEN‐(SGGS)2‐ALKBH5‐3xHA fusion protein (Figures 1e and S2), referred to as dcA. Additionally, we created catalytically dead ALKBH5 (ALKBH5H204A), referred to as dcdA, by mutagenizing the demethylation editor. Western blot analysis showed that both dcA and dcdA fusions were successfully expressed in tobacco leaf cells (Figure 1f).

Engineering the subcellular localization of the m6A editors

Endogenous MTA, MTB and ALKBH5 are confined to the nucleus to mediate the dynamic patterning of m6A modifications on mRNAs (Ruzicka et al., 2017; Zheng et al., 2013; Zhong et al., 2008). In this study, we added a NES to dcM and dcA fusions to extend their subcellular localization to the cytoplasm and sought to edit more mature mRNA transcripts in plant cells. To verify their subcellular localization, we replaced 3xHA with a GFP tag at the C‐terminus of the fusions (Figure S3) and transformed them into tobacco leaves. Confocal microscopy showed that the dCas13a‐GFP fusion without NES and the NES‐tagged dcA‐GFP fusion was localized in both the nucleus and cytoplasm, whereas the NES‐tagged dcM‐GFP fusion was localized only in the cytoplasm (Figure 2a). These results demonstrate that our m6A editors were successfully expressed and localized in the cytoplasm of plant cells.

Figure 2.

Figure 2

Open in a new tab

Validation of the programmable m6A editors in tobacco. (a) Subcellular localization of dCas13a, dcM and dcA fusion proteins in tobacco leaf cells. 35S::GFP was used as a control. Scale bar, 25 μm. (b) Schematic representation of the luciferase reporter construction and the regions targeted by the gRNAs. Orange rectangles indicate the m6A motifs (GGACU). Black arrows indicate the 5′‐3′ guide RNAs. gRNAs T1~5 were 10 nt, 50 nt, 100 nt, 250 nt and 500 nt to the nearest m6A motif site respectively. (c) m6A enrichment of the luciferase transcripts targeted by dcM editors. (d) m6A enrichment of the luciferase transcripts targeted by dcA editors. n = 3. Values are given as mean ± SD. *P < 0.05, **P < 0.01 and ***P < 0.001 using Student's t‐test.

Validation of the m6A editors on reporter transcripts in tobacco

To test the site‐specific m6A deposition and erasure of our editors on the individual transcripts in plants, we designed a reporter construct that contained the luciferase mRNA and eight m6A motifs (GGACU) in the 3′UTR of the luciferase sequence (Figure 2b). The luciferase assay showed that the reporter luciferase was well expressed with the m6A editors (Figure S4). To identify the editing window size of the dcM and dcA editors in plants, we designed guide RNAs (gRNAs) by principles as followed: (1) The gRNA should be reverse complement to targeted transcript RNAs. (2) The gRNA should be 28‐nucleotide (nt) length for dCas13a protein loading. (3) The gRNAs should be designed at a position near m6A site. Five gRNAs were designed at distinct positions to target the luciferase‐m6A motifs, with gRNA T1~5 at 10, 50, 100, 250 and 500 nt to the nearest m6A motif site, respectively (Figures 2b and S5a). To evaluate the methylation ability of the dcM editor, we co‐transformed reporter and methylation editors with different gRNAs in tobacco leaves. Western blot analysis showed that all dcM and dcdM editors were well expressed with mostly equal protein abundances in tobacco leaf cells (Figure S6a). Through quantitative PCR of RNA fragments immunoprecipitated with m6A antibodies (MeRIP‐qPCR), we measured m6A enrichment of the reporter transcripts. Our results showed that all the gRNA T1‐4 combined with the dcM editor significantly increased the m6A level of the reporter transcripts, whereas gRNA T5 showed no difference when compared to the non‐targeting gRNA and the counterpart gRNAs with inactive dcM editor (Figure 2c). The strongest methylation on the reporter transcripts was observed with gRNA T1, which was 10 nt from the nearest m6A site and resulted in ~3‐fold higher than non‐targeting gRNA (Figure 2c). These results suggest that our dcM editor could efficiently and specifically deposit m6A modifications on reporter transcripts, and the editing window was within 250 nt. We hypothesized that the dcM editor may have a higher editing efficiency when the gRNA is closer to the m6A site.

Next, we evaluated the demethylation ability of dcA editors in plants. To do this, we co‐transformed the luciferase reporter and demethylation editors with the same gRNA T1‐5 in tobacco leaves (Figures 2b and S5b). Similarly, we conducted western blot analysis for these editors and found that all the dcA and dcdA editors were well expressed in tobacco leaves with little difference (Figure S6b). MeRIP‐qPCR showed that all the gRNA T1‐3 with dcA editors significantly decreased m6A levels of the reporter transcripts when compared to the non‐targeting gRNA and the counterpart gRNAs with inactive dcdA editors, with gRNA T2 leading to ~80% demethylation (Figure 2d). The gRNA T4 with dcA editors showed no difference to the non‐target, but significantly lower m6A enrichment than with inactive dcdA editors, whereas gRNA T5 was unable to decrease the m6A levels of reporter transcripts (Figure 2d), suggesting that the effective editing window of the dcA editor was also within 250 nt. Collectively, these results suggest that our RNA methylation and demethylation editors based on the CRISPR/dCas13a system can efficiently and specifically deposit and remove m6A modifications on individual RNA transcripts with programmed guide RNAs near the target site in plant cells.

Validation of the m6A editors on the endogenous transcripts in Arabidopsis

To confirm the programmable m6A editing ability of our editors in endogenous transcripts, we sought to target known genes containing m6A modification in Arabidopsis. By carrying out a transcriptome‐wide MeRIP‐sequencing (MeRIP‐seq) in the flowers of Col‐0 and combining it with the reported m6A‐modified genes from previous studies (Anderson et al., 2018; Luo et al., 2014), we selected several m6A‐modified candidate genes which are essential for plant development. We located their m6A peak regions, and designed gRNAs for m6A editing (Figure S7). We first developed transgenic Arabidopsis overexpression of dcM editor with gRNAs targeting SHR, SCARECROW (SCR) for their important roles in root development (Helariutta et al., 2000), and targeting WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM) as a previous study revealed that m6A modifications of WUS and STM might be responsible for SAM over‐proliferation in FIP37 knockout mutants (Shen et al., 2016). We have obtained more than 30 independent transgenic T1 plants for each transgenic Arabidopsis, and randomly selected two independent T3 lines with comparable protein expression of m6A editors for further analysis. Western blot analysis showed that dcM and the negative control dcdM fusion proteins were well expressed in all overexpression lines. Since these genes are highly expressed in flowers, we conducted MeRIP‐qPCR on the flower tissues. We showed that overexpression of dcM with gRNA targeting SHR significantly increased the m6A enrichment of SHR transcripts by 1.86 ± 0.11 and 1.80 ± 0.09‐fold compared to the non‐target and the inactive dcdM, respectively, in two independent biological replicates (Figure 3b), indicating that the dcM editor could specifically instal m6A modification on the SHR transcript in Arabidopsis. For SCR, the dcM overexpression with gRNA showed 5.03 ± 0.43 and 3.20 ± 0.27‐fold higher m6A enrichment than with the non‐target and the inactive dcdM, respectively (Figure 3c). Additionally, targeting WUS and STM with dcM editors was shown to significantly induce m6A levels when compared to non‐target and inactive dcdM (Figure 3d,e), further indicating that the dcM editor has the capability of programmable m6A modification of endogenous transcripts in Arabidopsis.

Figure 3.

Figure 3

Open in a new tab

Validation of the programmable m6A editors in Arabidopsis. (a) Western blot analysis of the abundance of the dcM and dcdM editors in the flower tissues of transgenic Arabidopsis lines. Protein blots were developed with anti‐HA antibody. Ponceau staining served as loading control. (b–e) Relative m6A enrichment of endogenous SHR (b), SCR (c), WUS (d) and STM (e) transcripts targeted by dcM editors. (f) Western blot analysis of the abundance of the dcA and dcdA editors in the flower tissues of transgenic lines. Protein blots were developed with anti‐HA antibody. Ponceau staining served as loading control. (g–j) Relative m6A enrichment of endogenous SHR (g), SCR (h), PHB (i) and SOC1 (j) transcripts targeted by dcA editors. n = 3. Values are given as mean ± SD. Statistical significance is denoted by differing letters with P < 0.05 using Student's t‐test.

To test the targeted demethylation capability of the dcA editors on endogenous transcripts in Arabidopsis, we developed transgenic plants overexpressing the dcA editor with gRNAs targeting the m6A peak regions of SHR, SCR, PHB and SOC1. We confirmed dcA expression by western blot and found that both dcA and the negative control dcdA fusion proteins were well expressed in all transgenic lines (Figure 3f). The MeRIP‐qPCR showed that the overexpression of dcA significantly decreased the m6A level of SHR transcripts with 49.38 ± 6.28% and 56.92 ± 8.31% to non‐target and inactive dcdA, respectively, in flower tissues from two independent biological replicates (Figure 3g). Additionally, dcA overexpression with gRNA targeting SCR showed a significantly lower m6A enrichment, which was 11.76 ± 6.02% of the non‐target and 13.90 ± 7.20% of the inactive dcdA (Figure 3h), suggesting a high demethylation efficiency on SCR transcript. Furthermore, targeting two more m6A‐modified transcripts of PHB and SOC1 with dcA significantly reduced m6A enrichment compared to non‐target and inactive dcdA (Figure 3i,j). Taken together, these results demonstrated that with the programmable gRNAs, our dcM and dcA editors can successfully deposit and remove m6A modification on endogenous transcripts, respectively, in Arabidopsis.

Targeting SHR with the dcM editor induces plant growth

To date, there have been no reports of manipulating m6A levels in specific genes of interest to link phenotypic outcomes in any organism. Therefore, we sought to explore the possibility that editing the m6A levels of endogenous transcripts using our m6A editors can give rise to phenotypic changes in Arabidopsis. Interestingly, we found that overexpression of dcM with gRNA targeting SHR (dcM‐SHR) could significantly induce plant growth in many independent transgenic lines, but this result was not observed with non‐target (dcM‐non), inactive dcdM (dcdM‐SHR) and targeting SHR with dcA editors (Figure S8a). Additionally, we did not observe any obvious phenotypic changes when targeting other genes, such as SCR, WUS, STM, PHB and SOC1. These results suggest that the m6A modification on SHR transcripts may play an important role in plant growth. To further dissect the effects of methylation editing of SHR on phenotypic outcomes, we investigated the phenotypes of Arabidopsis transgenic line #1 plants at both vegetative and reproductive development stages. During vegetative development, dcM‐SHR seedlings showed a significantly longer root length than the wild type, Col‐0, at different developmental times after germination, whereas dcM‐non and dcdM‐SHR did not (Figure S9a,b). In addition, targeting SHR with both dcA and dcdA editors significantly decreased root length compared to Col‐0 (Figure S9c,d). At the 4‐week‐old stage, dcM‐SHR plants displayed an enlarged rosette and greater root developments (Figure 4a–c). By measuring the area of the largest rosette leaf in each seedling, we found that dcM‐SHR rosette leaves were significantly larger than those of Col‐0, dcM‐non, and dcdM‐SHR (Figure 4c,d). To confirm these, we also measured more independent T3 lines for each transgenic Arabidopsis, and found that the other four dcM‐SHR lines with comparable expression of editor proteins displayed increased leaf area compared to the control lines including dcM‐non and dcdM‐SHR (Figure S8b,c). Moreover, at the 8‐week‐old reproductive stage, the dcM‐SHR plants displayed increased plant biomass, with the plant height, aerial fresh and dry weights of dcM‐SHR being significantly higher than those of Col‐0, dcM‐non and dcdM‐SHR (Figure 4e–h). Consistent with the induced plant biomass, dcM‐SHR plants also showed significant increases in the number of siliques and total grain weight compared with Col‐0, dcM‐non and dcdM‐SHR (Figure 4i,j). In contrast, there was no significant difference in plant growth among Col‐0 and the transgenic Arabidopsis lines targeting SHR with dcA editors at the 4‐ and 8‐week‐old stages (Figure S10). Collectively, these results demonstrate that targeting the SHR transcript with our dcM editor to increase m6A levels in SHR can significantly promote plant growth and biomass. This suggests the potential to improve yield by targeting SHR orthologues with dcM editors in crops.

Figure 4.

Figure 4

Open in a new tab

Targeting SHR with dcM editor induces plant growth. (a–c) Rosette (a), root (b) and rosette leaf (c) phenotypes of Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR at 4 weeks old stage. Scale bar, 1 cm. (d) Comparison of rosette leaf areas. (e) Plant architecture of Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR at 8 weeks old stage. Scale bar, 5 cm. (f–j) Comparison of plant height (f), aerial fresh weight (g), aerial dry weight (h), number of siliques (i) and total grain weight (j) among Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR. More than 15 biological replicates for each genotypic line were measured for (d–j). (k) The mRNA lifetimes of SHR transcripts in 12‐day‐old Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR. 18S RNA was used as the internal control. TI, transcription inhibition. (l) The relative expression of SHR in 12‐day‐old Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR. (m) The mRNA lifetimes of SHR transcripts in 12‐day‐old Col‐0, dcA‐non, dcA‐SHR and dcdA‐SHR. (n) The relative expression of SHR in 12‐day‐old Col‐0, dcA‐non, dcA‐SHR and dcdA‐SHR. Three biological replicates for each genotypic line were measured for (k–n). Values are given as mean ± SD. Statistical significance is denoted by differing letters with P < 0.05 using Student's t‐test.

m6A editing of SHR regulates mRNA degradation to affect plant growth

The m6A modifications in Arabidopsis have been found to promote mRNA stabilization or mRNA degradation (Anderson et al., 2018; Shen et al., 2016; Wei et al., 2018). To confirm whether m6A modifications of SHR affect plant growth by post‐transcriptional regulation, we conducted transcription inhibition assays to detect the lifetimes of SHR transcripts in 12‐day‐old seedlings of different transgenic lines. We found that SHR transcripts were degraded more rapidly in dcM‐SHR when compared to the controls including Col‐0, dcM‐non, and dcdM‐SHR (Figure 4k). Consistent with this, the expression level of SHR was significantly reduced in dcM‐SHR lines (Figure 4m). In addition, we further detected mRNA stability in the demethylation editing plants and found that SHR transcripts were degraded more slowly in dcA‐SHR than those in Col‐0, dcA‐non and dcdA‐SHR, and the expression level of SHR was significantly increased in dcA‐SHR lines (Figure 4n,o). Moreover, the expression of SHR was significantly reduced in dcM‐SHR lines in different tissues such as root, shoot, leaf and flower compared to the controls including Col‐0, dcM‐non and dcdM‐SHR (Figure S11). These results indicated that the induced m6A levels of SHR by m6A editing with dcM editor can promote degradation of SHR transcripts to reduce its expression, and thus accelerate plant growth.

To further confirm the enhanced plant growth was attributed by the reduced expression of SHR, we developed SHR knockdown Arabidopsis (KD‐SHR) and compared the growth phenotype among Col‐0, KD‐SHR, 35S::SHR‐GFP overexpression line (OE‐SHR) and shr‐2 null mutant (Figure S12a–f). The qPCR analysis in leaves showed that SHR expression of OE‐SHR displayed ~15‐fold higher than that of Col‐0, whereas KD‐SHR#1~3 and #4~5 were ~0.5‐ and ~0.05‐fold SHR expression compared to Col‐0 respectively (Figure S12g). The shr‐2 mutant exhibited extreme growth defects with short shoot and small leaves, and OE‐SHR showed comparable growth and rosette leaf areas compared to Col‐0 (Figure S12a–c,f,h). Notably, KD‐SHR#1~3 that had moderately reduced SHR expression displayed enhanced plant growth and significantly increased rosette leaf area compared to Col‐0, similar to dcM‐SHR lines, whereas KD‐SHR#4~5 that had markedly reduced SHR expression exhibited short shoot and small leaves, just the same as shr‐2 (Figure S12d–f,h). Consistent with these, previous study also reported that the moderately reduced expression of SHR in Arabidopsis and PtSHR1 in Poplar could increase plant development (Wang et al., 2011). Together, these results demonstrate that the moderately reduced SHR is responsible for the enhanced plant growth in Arabidopsis. Meanwhile, the large amount loss of SHR expression or loss‐of‐function of SHR can lead to extreme growth defect, whereas the increase of SHR expression does not seriously affect plant growth in Arabidopsis.

Furthermore, we analysed the transcriptome profiles of Col‐0 and dcM‐SHR and identified a total of 588 differentially expressed genes, 379 of which were up‐regulated and 209 were down‐regulated (Figure S13a). Gene ontology (GO) analysis showed that some of the most significantly enriched terms were pollen tube development, multicellular organism process, regulation of growth from biological processes cation; transmembrane transporter activity, proton transmembrane transporter activity and hydrolysing O‐glycosyl compounds from molecular function (Figure S13b). KEGG pathway analysis showed that pentose and glucuronate interconversion, tryptophan metabolism, and nitrogen metabolism were among the most significantly enriched pathways (Figure S13c). Overall, these results suggest that targeting SHR with dcM editor can increase m6A levels and promote mRNA degradation to down‐regulate SHR transcripts, which thereby may cause changes of pathways in transporter activity, and tryptophan and nitrogen metabolism, resulting in induced plant growth and increased biomass in dcM‐SHR.

Specificity and off‐target of the dcM editor targeting SHR transcripts

To further verify that these phenotypic changes were caused by m6A editing with dcM editor targeting SHR instead of an off‐target effect on other transcripts, we performed a transcriptome‐wide MeRIP‐sequencing (MeRIP‐seq) in Arabidopsis lines. We first analysed the global features of m6A peaks in Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR from the MeRIP‐seq data. Most m6A peaks were distributed in protein‐coding sequences instead of tRNA, rRNA and non‐coding sequences (Figure S14a). The m6A peaks from protein‐coding transcripts were highly enriched in the CDS and stop codon (Figure S14b). Metagene analysis showed that the m6A peaks predominantly resided on the stop codon, which is consistent with previous reports (Figure S14c). These results suggest the high quality of the MeRIP‐seq data. Additionally, the m6A density peak in dcM‐non at the stop codon was higher than that in other groups (Figure S14c), suggesting a global increased m6A level in dcM‐non.

Next, we analysed the MeRIP‐seq data and detected the m6A peak in the editing region of SHR to evaluate the targeting specificity of the dcM editor. There was only one m6A peak in the 5′ CDS region of the SHR transcript, and this peak was higher in dcM‐SHR than in Col‐0, dcM‐non, and dcdM‐SHR (Figures 5a and S14d). These were consistent with the MeRIP‐qPCR results, further confirming the m6A editing capability of our dcM editor on endogenous transcripts. Moreover, to evaluate the off‐target effect of the dcM editor, we compared global m6A enrichment among Col‐0, dcM‐non, dcM‐SHR, and dcdM‐SHR. We found that dcM‐non showed significantly more global m6A enrichment than Col‐0 and dcM‐SHR, indicating a global increase in m6A levels, perhaps owing to the non‐targeting gRNA resulting in diffusion of dcM editor proteins to the global transcriptome (Figure 5b). The dcM‐SHR showed a significantly higher global m6A enrichment than Col‐0 but had no difference with dcdM‐SHR (Figure 5b). To further evaluate the off‐target effect, we conducted a pair‐wise comparison to display the different enriched m6A peaks by comparing dcM‐non, dcM‐SHR and dcdM‐SHR to Col‐0. We found that dcM‐non only showed 110 significantly up‐regulated and 171 down‐regulated m6A peaks, whereas dcdM‐SHR had 72 up‐regulated and 68 down‐regulated m6A peaks (Figure 5c). Similarly, dcM‐SHR showed 42 up‐regulated and 76 down‐regulated m6A peaks (Figure 5c). Fewer differential peaks of m6A enrichments suggested minimal off‐targeting in all editor lines. To further confirm phenotypic changes of dcM‐SHR is due to m6A editing of SHR transcripts instead of the off‐target effects, we performed GO analysis in the non‐specific genes with up‐ and down‐regulated m6A peaks in dcM‐SHR compared with Col‐0. The most significant GO terms were organelle fission and regulation of stomatal movement in genes with up‐ and down‐regulated m6A peaks, respectively, in dcM‐SHR compared to Col‐0 (Figure S15), neither of which were involved in pathways associated with plant growth. Together, these results suggest that m6A editing of SHR with dcM editor is responsible for the induced plant growth with minimal off‐target effects.

Figure 5.

Figure 5

Open in a new tab

Specificity and off‐target of dcM editor targeting SHR transcripts. (a) Genome browser view of m6A peak on SHR transcript in Col‐0, dcM‐non, dcM‐SHR and dcdM‐SHR, normalized by CPM (read counts per million). (b) Comparison of global m6A enrichment. The violin plots display the distribution of m6A peaks with the upper and lower whiskers indicating the max and min values, respectively, and the horizontal line in the middle of the box indicating the median value. ***P < 0.001 and ns indicates not significant, using Student's t‐test. (c) Volcano plots showing differential m6A peak enrichments between Col‐0 and dcM‐non, dcM‐SHR and dcdM‐SHR respectively (FC >1.5, P‐values <0.05). Red and blue points indicate up‐regulated and down‐regulated m6A peaks respectively. MeRIP‐seq analysis was performed with two independent biological replicates.

Discussion

Based on mutants of plant m6A writers, erasers and readers, a growing number of studies have revealed that m6A modification is widely involved in RNA processes and metabolism, including mRNA stability, alternative polyadenylation, translation and miRNA biogenesis, which play critical roles in plant development and stress responses. m6A‐associated epitranscriptomics has become a rapidly developing research field in plants. However, it has not been possible to dissect the function of m6A modification on a specific gene of interest because traditional biotechnology cannot manipulate the m6A level without changing the gene structure or transcription. In recent years, CRISPR/Cas systems have been extensively engineered into versatile tools for purposes of various genome and epigenome editing. The CRISPR‐associated Cas9 protein is an RNA‐guided DNA endonuclease that can recognize double‐stranded DNA for sequence‐specific cleavage. The recognition of Cas9 on DNA substrates requires a short DNA sequence of 5′‐NGG‐3′ (where ‘N’ is any nucleotide) known as the protospacer adjacent motif (PAM) and an 18 nt gRNA that is antisense to the opposite DNA strand. Thus, previous programmable editing systems based on CRISPR/Cas9 or CRISPR/dCas9 are mainly developed for genome editing, such as targeted knock‐out and knock‐in, base editor, DNA methylation, and histone modification editor, with target limitation because of the PAM sequence. Additionally, CRISPR/Cas9 system has also evolved to bind to single‐stranded RNA targets matching gRNA when the PAM is provided as an oligonucleotide (PAMmer) that hybridizes to the target RNA (O'Connell et al., 2014). Using specially designed gRNAs and PAMers, a previous study conducted m6A editing based on CRISPR/Cas9 system in mammalian cells (Liu et al., 2019). However, this system is difficult for m6A editing in plants for its complicated transformation of the oligonucleotide into cells. Unlike Cas9, CRISPR‐associated Cas13 protein can recognize the single‐stranded RNA and cuts the RNA sequence at a specific location with a 28 nt complementary gRNA assistance and its recognition does not require PAM in the sequence context. Recently, CRISPR/Cas13a has been shown to confer interference against RNA viruses in plants (Zhang et al., 2019). Therefore, compared to dCas9, the dCas13‐based system is more suitable for m6A editing in plants because of its easy design of target gRNA and simple constructions of the CRISPR/dCas13a system.

Based on the CRISPR/dCas13a system, we developed RNA methylation and demethylation editing tools to deposit and remove the m6A modification on specific transcripts in plants. The dcM RNA methylation tool was created by fusing the core domain of the MTA and MTB complex to the C‐terminus of dCas13a using a long flexible linker and combined with a programmable gRNA inserted into the same constructs, and the dcA RNA demethylation tool was created by fusing ALKBH5 to dCas13a with the same linker and gRNA (Figure 1). The NES tag was added to both the dcM and dcA editors to extend their subcellular localization to the cytoplasm of the plant cells. We validated the m6A editors in both tobacco and Arabidopsis, demonstrating that both the dcM and dcA editors could be successfully expressed and exhibited m6A editing activity with the programmable gRNA.

We confirmed that the effective editing window of dcM and dcA editors was within 250 nt, and gRNAs over 500 nt to the m6A site were unable to effectively carry out m6A editing (Figure 2c,d). The extensive editing window may be due to a very flexible accessibility of editors to the m6A site resulting from the complex three‐dimensional structure of target chromatin and the long protein linkers in editor proteins. In agreement with this, a previous study of m6A editing based on the CRISPR/dCas13 system also revealed a very extensive editing window in mammalian cells (Li et al., 2020a). This feature of the extensive editing window in dcM and dcA editors helps edit the endogenous transcripts in plants because currently a few model plants have been subjected to MeRIP‐seq, which provides the m6A peaks in terms of an interval region within about 200 nt. The exact m6A sites were difficult to obtain because of the technical restrictions of MeRIP‐seq. Thanks to the extensive editing window of the dcM and dcA editors, we performed m6A editing of endogenous transcripts by designing gRNAs within the m6A peak regions in Arabidopsis. Recently, novel methods such as m6A cross‐linking and immunoprecipitation sequencing (m6A‐CLIP/miCLIP‐seq) (Linder et al., 2015), nanopore direct RNA sequencing (Leger et al., 2021) and glyoxal and nitrite‐mediated deamination of unmethylated adenosine sequencing (GLORI‐seq) (Liu et al., 2023) can directly detect m6A modifications at single‐base resolution. We look forward to these new m6A sequencing techniques performed in plants, and thereby we can design more gRNAs and shorter linkers for targeted m6A editing of specific genes of interest to fine‐tune m6A levels to investigate its functions in plants. Additionally, we also observed that the inactive dcdM and dcdA editors exhibited a weak influence on the m6A enrichment of target transcripts compared to the non‐targets (Figures 2c,d and 3). We hypothesized that this might result from inactive editors binding to the target transcript, and affecting the proportion of m6A‐modified mRNA molecules and mRNA stability, thereby influencing the results of MeRIP‐qPCR, and perhaps the smaller Cas13 proteins, such as Cas13b and Cas13d, may be used to further eliminate this localization effect. Moreover, using MeRIP‐seq, we demonstrated that the dcM editor displayed specific methylation ability and limited off‐target effects (Figure 5). Collectively, these results suggest that our programmable m6A editing tools based on the CRISPR/Cas13a system can be easily applied to study m6A modification of specific genes in plants.

It is very common for the ectopic overexpression plants driven by Cauliflower Mosaic Virus (CaMV) 35S promoter to exhibit highly variable expression levels of the transgenic genes due to the co‐suppression phenomenon. In order to reduce this variation, we tended to select the transgenic Arabidopsis lines with comparable expression levels of the m6A editors. However, some lines such as SCR #1 and #2 with different overexpression levels of dcM editors showed no significant differences in relative m6A enrichment of SCR transcripts. Since m6A depositions are affected by many aspects, including the histone modification, transcript expression level, and total abundance of methyltransferase complex (Huang et al., 2019; Li et al., 2020b; Liu et al., 2020; Xu et al., 2021), we hypothesized that some degree of overexpressed dcM editors was able to sufficiently execute m6A editing on specific genes, while too higher abundance did not mean more editing efficiency. In agreement with these, there is no obvious correlation of editor abundance with the degree of m6A enrichment shown in previous studies of Cas13‐based m6A editing in mammalian cells (Chen et al., 2021; Li et al., 2020a; Wilson et al., 2020; Xia et al., 2021). Currently, we are trying to optimize the dcM and dcA editing system for stable expression in transgenic plants by using endogenous promoters such as promoters from Ubiquitin and Actin. Additionally, the off‐target effects are unavoidable in all CRISPR/Cas9‐ and CRISPR/Cas13‐based editing systems. In recent years, with the efforts of continuous optimization of Cas9 and Cas13 proteins and editing systems, more and more studies have been able to reduce the off‐target effects and promote editing efficiency. We are also trying to optimize the dCas13a‐based m6A editing system to promote editing efficiency and extend its application to other model plants.

Interestingly, we observed that targeting the SHR transcript with the dcM editor significantly induced plant growth with enlarged leaf and root size, increased plant height, aerial biomass, number of siliques and total grain weight (Figure 4). SHR is known to interact with SCR to maintain the growth of the root meristem, and the loss‐of‐function of SHR results in the disorganization of the quiescent centre and shortening of root length (Helariutta et al., 2000). In addition to the root, SHR can regulate leaf and shoot growth by controlling the differentiation of the bundle sheath and endodermis (Dhondt et al., 2010; Yoon et al., 2016). Previous studies have revealed that the reduced expression of SHR in Arabidopsis and PtSHR1 in Poplar could increase plant development (Wang et al., 2011). In agreement with this, our results confirmed that the moderately reduced SHR is responsible for the enhanced plant growth in SHR knockdown plants. Furthermore, we demonstrated that the increase in m6A levels induced by our dcM editor could promote degradation of SHR transcripts to reduce its expression, and thus enhance leaf size, plant height and biomass in dcM‐SHR plants. In addition, the increased aerial fresh weight and other parameters of inactive dcdM‐SHR plants may be also attributed by the slightly reduced SHR expression. Since m6A modification detected by MeRIP‐seq is not precise enough, we only obtained the m6A peak region in SHR transcripts, but the exact m6A site is still unclear. Therefore, we propose that future release of m6A single‐base resolution data will help design more gRNAs to fine‐tune m6A levels of SHR to control plant growth. Together, our study suggests that plant traits can be improved by manipulating m6A levels of specific gene transcripts using our editing tools. Similarly, a recent study reported that transgenic expression of FTO, which encodes human RNA demethylase, in rice and potato can enhance root growth, tiller bud formation, photosynthetic efficiency, and drought tolerance, resulting in a 50% increase in yield and biomass (Yu et al., 2021). These findings indicate that manipulating m6A levels is a promising strategy for improving plant traits. We propose that our programmable RNA methylation and demethylation tools have great potential for applications in future crop improvement.

Materials and methods

Plant materials and growth conditions

All wild‐type and transgenic lines used in this study were Arabidopsis thaliana ecotype Columbia (Col‐0) and its derivatives respectively. The transgenic 35S::SHR‐GFP line and shr‐2 null mutant (CS2972) came from the previous study of Shuang Wu (Wu et al., 2014). Seeds were surface‐sterilized with 75% alcohol for 1 min and 30% sodium hypochlorite for 10 min, before being rinsed five times with sterile distilled water. Seeds were then soaked in sterile water at 4 °C for 2 days. The seeds were finally grown on half‐strength Murashige and Skoog (MS) medium under a long‐day photoperiod (16 h light/8 h dark, at 22 °C) for ~10 days. The seedlings were then transferred to soil in growth chambers under the same conditions. The seeds of Nicotiana benthamiana were grown and transplanted into the soil under the same long‐day photoperiod (16 h light/8 h darkness, at 22 °C) in growth chambers.

Plasmid construction

Plant codon‐optimized LshCas13a was obtained from a previous study by Jiang Zhang (Zhan et al., 2019). We mutated four amino acids, R597A, H602A, R1278A and H1283A, in the HEPN domain of Cas13a to generate catalytically dead Cas13a (dCas13a). The pCR11 plasmid backbone containing the AtU6 driving guide RNA for LshCas13a came from the previous study of Guohui Zhou (Zhang et al., 2019). Sequences of the (GGS)10 and (SGGS)2‐XTEN‐(SGGS)2 linkers were artificially synthesized. We amplified the sequences of ΔMTA 446–667 and ΔMTB 562–876 from the cDNA and cloned them with dCas13, linkers, NES‐tag, and HA‐tag into the pCR11 plasmid backbone to generate a dcM methylation editor. D482A, a mutagenesis in ΔMTA446–667, was created to generate an inactive dcdM editor. Human ALKBH5 came from the previous study by Hongsheng Wang (Li et al., 2020a). We cloned ALKBH5 and ALKBH5 H204A with dCas13, (SGGS)2‐XTEN‐(SGGS)2 linker, NES‐tag, and HA‐tag into the pCR11 plasmid backbone to generate the dcA and dcdA editors. We also cloned dCas13a with an HA‐tag into the pCR11 plasmid backbone to generate dCas13‐HA as a control. For subcellular localization analysis in tobacco, the constructs were generated by replacing the HA‐tag with a GFP tag. The guide RNAs were cloned via a golden gate into the editor construction bearing the LshCas13a crisp RNA backbone under the Arabidopsis U6 promoter. Eight m6A motif sequence was artificially synthesized and cloned into the 3′‐UTR of the luciferase sequence driven by the 35S promoter. The 201‐bp sequence from SHR cDNA and its reverse complementary sequence were artificially synthesized, and cloned into RNAi vector to develop the SHR knockdown construct. All primers and synthesized sequences used in this study are listed in Tables S1–S3.

Plant transformation

All constructs were introduced into Agrobacterium tumefaciens strain GV3101. For the transient transformation of Nicotiana benthamiana leaves, strains harbouring different expression constructs were resuspended and adjusted to OD600 = 1.0 in activating solution (10 mM MES pH 5.6, 10 mM MgCl2 and 150 μM acetosyringone), and left to rest at room temperature for 3 h. Then, the activated strains were mixed in 1:1 or 1:1:1 with the pSou‐p19 strain, infiltrated into 4‐week‐old tobacco leaves and cultivated under normal conditions before observation. For the transformation of Arabidopsis, strains harbouring different expression constructs were resuspended and adjusted to OD600 = 1.0 in 5% sucrose solution, and introduced into 6‐week‐old Arabidopsis plants using the floral dip method as previously described (Zhang et al., 2006). T3 homozygous transgenic plants were used for m6A detection and phenotypic observations.

Western blot

The samples collected from tobacco leaves 3 days after transformation were ground into a fine powder using liquid nitrogen and homogenized in lysis buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM MgCl2, 10% glycerol, 0.1% NP‐40, 0.5 mM DTT, 1 mM PMSF and 1× Protease Inhibitor Cocktail (Roche)). The leaf lysates were imbibed on ice for 30 min and centrifuged at 12 000 g at 4 °C for 30 min. The supernatant was extracted, mixed with 5× SDS loading buffer and heated at 98 °C for 10 min. The protein mixtures were loaded onto a 4%–12% PAGE protein gel for electrophoresis at a constant voltage of 120 V until the front dye reached the bottom of the gel. Then, the protein samples were transferred to 0.45 μm PVDF membranes (Merck) using the Trans‐Blot SD semi‐dry transfer cell (BioRad) and blocked in 5% non‐fat milk in TBST, followed by incubation with anti‐HA and goat anti‐mouse IgG antibodies (Abclonal, China) for western blotting, according to the manufacturer's instructions.

MeRIP‐qPCR

Total RNA from tobacco leaves and Arabidopsis flowers was isolated using TRIzol reagent (Invitrogen). The following m6A MeRIP protocol was based on a previous study (Zeng et al., 2018) with minor modifications: 2 μg total RNA was added to 300 μL IP buffer (150 mM NaCl, 10 mM Tris–HCl [pH 7.5], 0.1% IGEPAL CA‐630 in nuclease‐free DEPC‐treated H2O), incubated with 1 μg anti‐m6A antibody (Synaptic Systems) and tumbled at 4 °C for 2 h. Then, the reaction mixture was incubated with protein A/G magnetic beads (MedChemExpress) and tumbled at 4 °C for 1 h. The reaction mixture was then washed twice with IP buffer, twice with low‐salt IP buffer (50 mM NaCl, 10 mM Tris–HCl [pH 7.5], 0.1% IGEPAL CA‐630 in nuclease‐free DEPC‐treated H2O), and twice with high‐salt IP buffer (500 mM NaCl, 10 mM Tris–HCl [pH 7.5], 0.1% IGEPAL CA‐630 in nuclease‐free DEPC‐treated H2O) at 4 °C for 10 min each. After extensive washing, the m6A‐enriched RNA was purified using TRIzol reagent with glycol blue co‐precipitant (Invitrogen). All purified m6A‐enriched RNA and 1 μg total RNA were reverse‐transcribed into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit with a gDNA wiper (Vazyme, China). Real‐time quantitative PCR was performed on a CFX connect Real‐Time system (BioRad) using ChamQ Universal SYBR qPCR Master Mix (Vazyme). Relative m6A enrichment was calculated as previously described (Zeng et al., 2018): ΔCt = Ct of the target gene in the IP sample – Ct of the target gene in the input sample, ΔΔCt = ΔCt of the treated sample – ΔCt of the control sample, and relative m6A enrichment = 2−ΔΔCt. Three biological replicates were performed with the non‐target used as a control reference to normalize m6A enrichment for MeRIP‐qPCR, and ACTIN2 was used as an internal reference to normalize gene expression for RT‐qPCR.

mRNA stability measurements

An mRNA stability measurement assay was based on previously described protocols (Wei et al., 2018). Briefly, 12‐day‐old Col‐0, dcM‐non, dcM‐SHR, dcdM‐SHR, dcA‐non, dcA‐SHR and dcdA‐SHR Arabidopsis seedlings grown on 1/2 MS medium were transferred to 5‐cm Petri dishes containing 1/2 MS liquid medium and incubated for 30 min. Then, 0.2 mM actinomycin D was added to the buffer to inhibit transcription. Samples were collected at 1 h after the transcription inhibitor was added and were referred to as 0 h samples. The 2, 4, 6, and 8 h samples were collected and immediately frozen in liquid nitrogen. Total RNA from these samples was isolated using TRIzol reagent (Invitrogen). cDNA was generated with HiScript III 1st Strand cDNA Synthesis Kit with a gDNA wiper (Vazyme, China). Real‐time quantitative PCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme). 18S RNA was used as the internal control.

MeRIP‐seq

MeRIP‐seq was based on previously described protocols (Dominissini et al., 2013; Zeng et al., 2018) with minor modifications. Total RNA from Arabidopsis flowers was isolated using TRIzol reagent, and 100 μg of total RNA was polyA‐selected to obtain 2 μg of intact mRNA using a Dynabeads mRNA Purification Kit (Invitrogen). All isolated mRNA were chemically fragmented using the NEBNext Magnesium RNA Fragmentation Module (New England Biolabs, UK). We aliquoted ~300 ng of fragmented mRNA for use as input for MeRIP‐seq. The remaining fragmented mRNA was added to a 500 μL IP buffer, incubated with 5 μg anti‐m6A antibody (Synaptic Systems), and 5 μL of RNasin Plus RNase Inhibitor (Promega), and tumbled at 4 °C for 2 h. Then, the reaction mixture was incubated with protein A/G magnetic beads (MedChemExpress) and tumbled at 4 °C for 1 h. Then, the reaction mixture was washed twice with IP buffer, twice with low‐salt IP buffer, and twice with high‐salt IP buffer at 4 °C for 10 min for each. After extensive washing, the m6A‐enriched RNA was purified using TRIzol reagent with glycol blue co‐precipitant (Invitrogen). All input and IP RNA were used for RNA library construction using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs) according to the manufacturer's instructions. All RNA libraries were sequenced (Paired‐end 150 nt) on an Illumina NovaSeq 6000 platform (Novogene, China).

Data analysis

The MeRIP‐seq raw data were processed using in‐house Perl scripts by removing the 5′ adapter, reads with more than 10% N, and reads with more than 50% nucleotides with Qphred ≤5. Clean reads were then mapped to the TAIR10 Arabidopsis genome using HISAT2 (Kim et al., 2019). Enriched m6A peaks were identified by MACS2 (Zhang et al., 2008) using the corresponding input library as a control. Peak annotation and metagene analysis of m6A distribution in transcripts were accomplished using RNAmod (Liu and Gregory, 2019). m6A peak differential analysis was performed using the R package ‘MeTDiff’ (Cui et al., 2018). The input libraries were used for transcriptome analysis, and differential expression analysis (FC >1.5, P‐value <0.05) was conducted using CUFFLINKS (Trapnell et al., 2010). GO and KEGG analyses were performed using the R package ‘clusterProfiler’ (Wu et al., 2021).

Accession numbers

Gene sequence data for this study can be found in the Arabidopsis Genome Initiative under the following accession numbers: MTA (AT4G10760), MTB (AT4G09980), SHR (AT4G37650), SCR (AT3G54220), WUS (AT2G17950), STM (AT1G62360), PHB (AT2G34710), SOC1 (AT2G45660) and ACTIN2 (AT3G18780).

Conflict of interest

The authors declare no competing interest.

Funding information

This study was supported by the National Natural Science Foundation of China (32188102, 32100289), Special Funds for Science Technology Innovation and Industrial Development of Shenzhen Dapeng New District (KJYF202001‐01), the Chinese Postdoctoral Science Foundation (2021M703539), and Youth Innovation of the Chinese Academy of Agricultural Sciences (Y20230C36).

Author contributions

This research was conceived by Q.Q., L. S., and Q. F. Experiments were conducted by C. S., W. Z., X. L., H. Z., X. L., and G. F. Data analyses were performed by C. S., X. L., and H. Z., C. S. and W. Z. wrote the manuscript, and all authors contributed to editing the manuscript.

Supporting information

Figure S1 The amino acid sequence of dcM/dcdM editor.

Figure S2 The amino acid sequence of dcA/dcdA editor.

Figure S3 Schematic representation of the constructions for subcellular localization in tobacco cells.

Figure S4 Luciferase assay of reporter transcript containing m6A motifs in the 3′UTR in tobacco leaves.

Figure S5 Schematic representation of the constructions for validation of the m6A editors in tobacco cells.

Figure S6 The protein abundance analysis of the editors in tobacco leaf cells.

Figure S7 Genome browser view of m6A peaks and designed gRNAs in candidate target genes for m6A editing.

Figure S8 The phenotype of transgenic overexpression of m6A editors with gRNA targeting SHR.

Figure S9 Root phenotype of Arabidopsis seedlings overexpressing dcM and dcA editors targeting SHR transcript.

Figure S10 The phenotype of transgenic overexpression of demethylation editors with gRNA targeting SHR.

Figure S11 The expression pattern of SHR in different genotypic plants.

Figure S12 The phenotype of transgenic SHR overexpression and knockdown plants.

Figure S13 Differentially expressed genes analysis between Col‐0 and dcM‐SHR.

Figure S14 Peak annotation and m6A distribution analysis with MeRIP‐seq data.

Figure S15 The GO enrichment analysis of the non‐specific genes with up‐ and down‐regulated m6A peaks in dcM‐SHR compared with Col‐0.

Table S1 Primers used in this study.

Table S2 dLshCas13a guide RNA sequences used in this study.

Table S3 Synthesized oligonucleotide sequences used in this study.

PBI-22-1867-s001.pdf (1.1MB, pdf)

Acknowledgements

We thank Prof. Jiang Zhang at Hubei University for providing us with the plant codon‐optimized LshCas13a plasmid. We thank Prof. Guohui Zhou at the South China Agricultural University for providing us with the pCR11 plasmid backbone. We thank Prof. Hongsheng Wang at Sun Yat‐sen University for providing the human ALKBH5 plasmid. We thank Prof. Shuang Wu at Fujian Agriculture and Forestry University for providing the 35S::SHR‐GFP and shr‐2 null mutant Arabidopsis plants. We thank the members of the Shang Lab for their helpful discussions.

Contributor Information

Qili Fei, Email: feiqili@live.cn.

Qian Qian, Email: qianqian188@hotmail.com.

Lianguang Shang, Email: shanglianguang@caas.cn.

Data availability statement

The MeRIP sequencing data generated in this study are available in the Gene Expression Omnibus (GEO) as GSE221143. Other data generated during this study are included in this article and the Supporting Information files.

References

  1. Abudayyeh, O.O. , Gootenberg, J.S. , Essletzbichler, P. , Han, S. , Joung, J. , Belanto, J.J. , Verdine, V. et al. (2017) RNA targeting with CRISPR‐Cas13. Nature, 550, 280–284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Abudayyeh, O.O. , Gootenberg, J.S. , Konermann, S. , Joung, J. , Slaymaker, I.M. , Cox, D.B. , Shmakov, S. et al. (2016) C2c2 is a single‐component programmable RNA‐guided RNA‐targeting CRISPR effector. Science, 353, aaf5573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Aman, R. , Ali, Z. , Butt, H. , Mahas, A. , Aljedaani, F. , Khan, M.Z. , Ding, S. et al. (2018) RNA virus interference via CRISPR/Cas13a system in plants. Genome Biol. 19, 1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson, S.J. , Kramer, M.C. , Gosai, S.J. , Yu, X. , Vandivier, L.E. , Nelson, A.D.L. , Anderson, Z.D. et al. (2018) N(6)‐methyladenosine inhibits local ribonucleolytic cleavage to stabilize mRNAs in Arabidopsis . Cell Rep. 25, 1146–1157.e1143. [DOI] [PubMed] [Google Scholar]
  5. Boccaletto, P. , Stefaniak, F. , Ray, A. , Cappannini, A. , Mukherjee, S. , Purta, E. , Kurkowska, M. et al. (2022) MODOMICS: a database of RNA modification pathways. 2021 update. Nucleic Acids Res. 50, D231–D235. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bodi, Z. , Zhong, S. , Mehra, S. , Song, J. , Graham, N. , Li, H. , May, S. et al. (2012) Adenosine methylation in Arabidopsis mRNA is associated with the 3′ end and reduced levels cause developmental defects. Front. Plant Sci. 3, 48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bokar, J.A. , Shambaugh, M.E. , Polayes, D. , Matera, A.G. and Rottman, F.M. (1997) Purification and cDNA cloning of the AdoMet‐binding subunit of the human mRNA (N6‐adenosine)‐methyltransferase. RNA, 3, 1233–1247. [PMC free article] [PubMed] [Google Scholar]
  8. Chen, X. , Zhao, Q. , Zhao, Y.L. , Chai, G.S. , Cheng, W. , Zhao, Z. , Wang, J. et al. (2021) Targeted RNA N(6)‐methyladenosine demethylation controls cell fate transition in human pluripotent stem cells. Adv. Sci. (Weinh). 8, e2003902. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cui, X. , Zhang, L. , Meng, J. , Rao, M.K. , Chen, Y. and Huang, Y. (2018) MeTDiff: a novel differential RNA methylation analysis for MeRIP‐seq data. IEEE/ACM Trans. Comput. Biol. Bioinform. 15, 526–534. [DOI] [PubMed] [Google Scholar]
  10. Dhondt, S. , Coppens, F. , De Winter, F. , Swarup, K. , Merks, R.M. , Inze, D. , Bennett, M.J. et al. (2010) SHORT‐ROOT and SCARECROW regulate leaf growth in Arabidopsis by stimulating S‐phase progression of the cell cycle. Plant Physiol. 154, 1183–1195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dominissini, D. , Moshitch‐Moshkovitz, S. , Salmon‐Divon, M. , Amariglio, N. and Rechavi, G. (2013) Transcriptome‐wide mapping of N(6)‐methyladenosine by m(6)A‐seq based on immunocapturing and massively parallel sequencing. Nat. Protoc. 8, 176–189. [DOI] [PubMed] [Google Scholar]
  12. Duan, H.C. , Wei, L.H. , Zhang, C. , Wang, Y. , Chen, L. , Lu, Z. , Chen, P.R. et al. (2017) ALKBH10B Is an RNA N(6)‐methyladenosine demethylase affecting Arabidopsis floral transition. Plant Cell, 29, 2995–3011. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. East‐Seletsky, A. , O'Connell, M.R. , Knight, S.C. , Burstein, D. , Cate, J.H. , Tjian, R. and Doudna, J.A. (2016) Two distinct RNase activities of CRISPR‐C2c2 enable guide‐RNA processing and RNA detection. Nature, 538, 270–273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Helariutta, Y. , Fukaki, H. , Wysocka‐Diller, J. , Nakajima, K. , Jung, J. , Sena, G. , Hauser, M.T. et al. (2000) The SHORT‐ROOT gene controls radial patterning of the Arabidopsis root through radial signaling. Cell, 101, 555–567. [DOI] [PubMed] [Google Scholar]
  15. Hou, Y. , Sun, J. , Wu, B. , Gao, Y. , Nie, H. , Nie, Z. , Quan, S. et al. (2021) CPSF30‐L‐mediated recognition of mRNA m(6)A modification controls alternative polyadenylation of nitrate signaling‐related gene transcripts in Arabidopsis . Mol. Plant, 14, 688–699. [DOI] [PubMed] [Google Scholar]
  16. Huang, H. , Weng, H. , Zhou, K. , Wu, T. , Zhao, B.S. , Sun, M. , Chen, Z. et al. (2019) Histone H3 trimethylation at lysine 36 guides m(6)A RNA modification co‐transcriptionally. Nature, 567, 414–419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jia, G. , Fu, Y. , Zhao, X. , Dai, Q. , Zheng, G. , Yang, Y. , Yi, C. et al. (2011) N6‐methyladenosine in nuclear RNA is a major substrate of the obesity‐associated FTO. Nat. Chem. Biol. 7, 885–887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kasowitz, S.D. , Ma, J. , Anderson, S.J. , Leu, N.A. , Xu, Y. , Gregory, B.D. , Schultz, R.M. et al. (2018) Nuclear m6A reader YTHDC1 regulates alternative polyadenylation and splicing during mouse oocyte development. PLoS Genet. 14, e1007412. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kim, D. , Paggi, J.M. , Park, C. , Bennett, C. and Salzberg, S.L. (2019) Graph‐based genome alignment and genotyping with HISAT2 and HISAT‐genotype. Nat. Biotechnol. 37, 907–915. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Komor, A.C. , Badran, A.H. and Liu, D.R. (2017) CRISPR‐based technologies for the manipulation of eukaryotic genomes. Cell, 168, 20–36. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kordys, M. , Sen, R. and Warkocki, Z. (2022) Applications of the versatile CRISPR‐Cas13 RNA targeting system. Wiley Interdiscip. Rev. RNA, 13, e1694. [DOI] [PubMed] [Google Scholar]
  22. Leger, A. , Amaral, P.P. , Pandolfini, L. , Capitanchik, C. , Capraro, F. , Miano, V. , Migliori, V. et al. (2021) RNA modifications detection by comparative Nanopore direct RNA sequencing. Nat. Commun. 12, 7198. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Li, J. , Chen, Z. , Chen, F. , Xie, G. , Ling, Y. , Peng, Y. , Lin, Y. et al. (2020a) Targeted mRNA demethylation using an engineered dCas13b‐ALKBH5 fusion protein. Nucleic Acids Res. 48, 5684–5694. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Li, Y. , Xia, L. , Tan, K. , Ye, X. , Zuo, Z. , Li, M. , Xiao, R. et al. (2020b) N(6)‐methyladenosine co‐transcriptionally directs the demethylation of histone H3K9me2. Nat. Genet. 52, 870–877. [DOI] [PubMed] [Google Scholar]
  25. Linder, B. , Grozhik, A.V. , Olarerin‐George, A.O. , Meydan, C. , Mason, C.E. and Jaffrey, S.R. (2015) Single‐nucleotide‐resolution mapping of m6A and m6Am throughout the transcriptome. Nat. Methods, 12, 767–772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liu, C. , Sun, H. , Yi, Y. , Shen, W. , Li, K. , Xiao, Y. , Li, F. et al. (2023) Absolute quantification of single‐base m(6)A methylation in the mammalian transcriptome using GLORI. Nat. Biotechnol. 41, 355–366. [DOI] [PubMed] [Google Scholar]
  27. Liu, J. , Dou, X. , Chen, C. , Chen, C. , Liu, C. , Xu, M.M. , Zhao, S. et al. (2020) N (6)‐methyladenosine of chromosome‐associated regulatory RNA regulates chromatin state and transcription. Science, 367, 580–586. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Liu, J. , Yue, Y. , Han, D. , Wang, X. , Fu, Y. , Zhang, L. , Jia, G. et al. (2014) A METTL3‐METTL14 complex mediates mammalian nuclear RNA N6‐adenosine methylation. Nat. Chem. Biol. 10, 93–95. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Liu, Q. and Gregory, R.I. (2019) RNAmod: an integrated system for the annotation of mRNA modifications. Nucleic Acids Res. 47, W548–W555. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Liu, X.M. , Zhou, J. , Mao, Y. , Ji, Q. and Qian, S.B. (2019) Programmable RNA N(6)‐methyladenosine editing by CRISPR‐Cas9 conjugates. Nat. Chem. Biol. 15, 865–871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Luo, G.Z. , MacQueen, A. , Zheng, G. , Duan, H. , Dore, L.C. , Lu, Z. , Liu, J. et al. (2014) Unique features of the m6A methylome in Arabidopsis thaliana . Nat. Commun. 5, 5630. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Martinez‐Perez, M. , Aparicio, F. , Lopez‐Gresa, M.P. , Belles, J.M. , Sanchez‐Navarro, J.A. and Pallas, V. (2017) Arabidopsis m(6)A demethylase activity modulates viral infection of a plant virus and the m(6)A abundance in its genomic RNAs. Proc. Natl. Acad. Sci. USA, 114, 10755–10760. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Meyer, K.D. , Patil, D.P. , Zhou, J. , Zinoviev, A. , Skabkin, M.A. , Elemento, O. , Pestova, T.V. et al. (2015) 5′ UTR m(6)A promotes cap‐independent translation. Cell, 163, 999–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. O'Connell, M.R. , Oakes, B.L. , Sternberg, S.H. , East‐Seletsky, A. , Kaplan, M. and Doudna, J.A. (2014) Programmable RNA recognition and cleavage by CRISPR/Cas9. Nature, 516, 263–266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Ping, X.L. , Sun, B.F. , Wang, L. , Xiao, W. , Yang, X. , Wang, W.J. , Adhikari, S. et al. (2014) Mammalian WTAP is a regulatory subunit of the RNA N6‐methyladenosine methyltransferase. Cell Res. 24, 177–189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Roundtree, I.A. , Luo, G.Z. , Zhang, Z. , Wang, X. , Zhou, T. , Cui, Y. , Sha, J. et al. (2017) YTHDC1 mediates nuclear export of N(6)‐methyladenosine methylated mRNAs. elife, 6, e31311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Ruzicka, K. , Zhang, M. , Campilho, A. , Bodi, Z. , Kashif, M. , Saleh, M. , Eeckhout, D. et al. (2017) Identification of factors required for m(6) A mRNA methylation in Arabidopsis reveals a role for the conserved E3 ubiquitin ligase HAKAI. New Phytol. 215, 157–172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Schellenberger, V. , Wang, C.W. , Geething, N.C. , Spink, B.J. , Campbell, A. , To, W. , Scholle, M.D. et al. (2009) A recombinant polypeptide extends the in vivo half‐life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186–1190. [DOI] [PubMed] [Google Scholar]
  39. Schwartz, S. , Mumbach, M.R. , Jovanovic, M. , Wang, T. , Maciag, K. , Bushkin, G.G. , Mertins, P. et al. (2014) Perturbation of m6A writers reveals two distinct classes of mRNA methylation at internal and 5′ sites. Cell Rep. 8, 284–296. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Scutenaire, J. , Deragon, J.M. , Jean, V. , Benhamed, M. , Raynaud, C. , Favory, J.J. , Merret, R. et al. (2018) The YTH domain protein ECT2 is an m(6)A reader required for normal trichome branching in Arabidopsis . Plant Cell, 30, 986–1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Shen, L. , Liang, Z. , Gu, X. , Chen, Y. , Teo, Z.W. , Hou, X. , Cai, W.M. et al. (2016) N(6)‐methyladenosine RNA modification regulates shoot stem cell fate in Arabidopsis . Dev. Cell, 38, 186–200. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Song, P. , Yang, J. , Wang, C. , Lu, Q. , Shi, L. , Tayier, S. and Jia, G. (2021) Arabidopsis N(6)‐methyladenosine reader CPSF30‐L recognizes FUE signals to control polyadenylation site choice in liquid‐like nuclear bodies. Mol. Plant, 14, 571–587. [DOI] [PubMed] [Google Scholar]
  43. Tang, J. , Yang, J. , Duan, H. and Jia, G. (2021) ALKBH10B, an mRNA m(6)A demethylase, modulates ABA response during seed germination in Arabidopsis . Front. Plant Sci. 12, 712713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Trapnell, C. , Williams, B.A. , Pertea, G. , Mortazavi, A. , Kwan, G. , van Baren, M.J. , Salzberg, S.L. et al. (2010) Transcript assembly and quantification by RNA‐Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat. Biotechnol. 28, 511–515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang, J. , Andersson‐Gunneras, S. , Gaboreanu, I. , Hertzberg, M. , Tucker, M.R. , Zheng, B. , Lesniewska, J. et al. (2011) Reduced expression of the SHORT‐ROOT gene increases the rates of growth and development in hybrid poplar and Arabidopsis . PLoS One, 6, e28878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wang, X. , Lu, Z. , Gomez, A. , Hon, G.C. , Yue, Y. , Han, D. , Fu, Y. et al. (2014) N6‐methyladenosine‐dependent regulation of messenger RNA stability. Nature, 505, 117–120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Wang, X. , Zhao, B.S. , Roundtree, I.A. , Lu, Z. , Han, D. , Ma, H. , Weng, X. et al. (2015) N(6)‐methyladenosine modulates messenger RNA translation efficiency. Cell, 161, 1388–1399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wei, L.H. , Song, P. , Wang, Y. , Lu, Z. , Tang, Q. , Yu, Q. , Xiao, Y. et al. (2018) The m(6)A reader ECT2 controls trichome morphology by affecting mRNA stability in Arabidopsis . Plant Cell, 30, 968–985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Wilson, C. , Chen, P.J. , Miao, Z. and Liu, D.R. (2020) Programmable m(6)A modification of cellular RNAs with a Cas13‐directed methyltransferase. Nat. Biotechnol. 38, 1431–1440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wu, S. , Lee, C.M. , Hayashi, T. , Price, S. , Divol, F. , Henry, S. , Pauluzzi, G. et al. (2014) A plausible mechanism, based upon Short‐Root movement, for regulating the number of cortex cell layers in roots. Proc. Natl. Acad. Sci. USA, 111, 16184–16189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Wu, T. , Hu, E. , Xu, S. , Chen, M. , Guo, P. , Dai, Z. , Feng, T. et al. (2021) clusterProfiler 4.0: a universal enrichment tool for interpreting omics data. Innovation (Camb). 2, 100141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Xia, Z. , Tang, M. , Ma, J. , Zhang, H. , Gimple, R.C. , Prager, B.C. , Tang, H. et al. (2021) Epitranscriptomic editing of the RNA N6‐methyladenosine modification by dCasRx conjugated methyltransferase and demethylase. Nucleic Acids Res. 49, 7361–7374. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Xiao, W. , Adhikari, S. , Dahal, U. , Chen, Y.S. , Hao, Y.J. , Sun, B.F. , Sun, H.Y. et al. (2016) Nuclear m(6)A reader YTHDC1 regulates mRNA splicing. Mol. Cell, 61, 507–519. [DOI] [PubMed] [Google Scholar]
  54. Xu, W. , Li, J. , He, C. , Wen, J. , Ma, H. , Rong, B. , Diao, J. et al. (2021) METTL3 regulates heterochromatin in mouse embryonic stem cells. Nature, 591, 317–321. [DOI] [PubMed] [Google Scholar]
  55. Yoon, E.K. , Dhar, S. , Lee, M.H. , Song, J.H. , Lee, S.A. , Kim, G. , Jang, S. et al. (2016) Conservation and diversification of the SHR‐SCR‐SCL23 regulatory network in the development of the functional endodermis in Arabidopsis shoots. Mol. Plant, 9, 1197–1209. [DOI] [PubMed] [Google Scholar]
  56. Yu, Q. , Liu, S. , Yu, L. , Xiao, Y. , Zhang, S. , Wang, X. , Xu, Y. et al. (2021) RNA demethylation increases the yield and biomass of rice and potato plants in field trials. Nat. Biotechnol. 39, 1581–1588. [DOI] [PubMed] [Google Scholar]
  57. Yue, H. , Nie, X. , Yan, Z. and Weining, S. (2019) N6‐methyladenosine regulatory machinery in plants: composition, function and evolution. Plant Biotechnol. J. 17, 1194–1208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Yue, Y. , Liu, J. , Cui, X. , Cao, J. , Luo, G. , Zhang, Z. , Cheng, T. et al. (2018) VIRMA mediates preferential m(6)A mRNA methylation in 3′UTR and near stop codon and associates with alternative polyadenylation. Cell Discov. 4, 10. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Zeng, Y. , Wang, S. , Gao, S. , Soares, F. , Ahmed, M. , Guo, H. , Wang, M. et al. (2018) Refined RIP‐seq protocol for epitranscriptome analysis with low input materials. PLoS Biol. 16, e2006092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhan, X. , Zhang, F. , Zhong, Z. , Chen, R. , Wang, Y. , Chang, L. , Bock, R. et al. (2019) Generation of virus‐resistant potato plants by RNA genome targeting. Plant Biotechnol. J. 17, 1814–1822. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Zhang, T. , Zhao, Y. , Ye, J. , Cao, X. , Xu, C. , Chen, B. , An, H. et al. (2019) Establishing CRISPR/Cas13a immune system conferring RNA virus resistance in both dicot and monocot plants. Plant Biotechnol. J. 17, 1185–1187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zhang, X. , Henriques, R. , Lin, S.S. , Niu, Q.W. and Chua, N.H. (2006) Agrobacterium‐mediated transformation of Arabidopsis thaliana using the floral dip method. Nat. Protoc. 1, 641–646. [DOI] [PubMed] [Google Scholar]
  63. Zhang, Y. , Liu, T. , Meyer, C.A. , Eeckhoute, J. , Johnson, D.S. , Bernstein, B.E. , Nusbaum, C. et al. (2008) Model‐based analysis of ChIP‐Seq (MACS). Genome Biol. 9, R137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Zhao, B.S. , Roundtree, I.A. and He, C. (2017) Post‐transcriptional gene regulation by mRNA modifications. Nat. Rev. Mol. Cell Biol. 18, 31–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Zheng, G. , Dahl, J.A. , Niu, Y. , Fedorcsak, P. , Huang, C.M. , Li, C.J. , Vagbo, C.B. et al. (2013) ALKBH5 is a mammalian RNA demethylase that impacts RNA metabolism and mouse fertility. Mol. Cell, 49, 18–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  66. Zhong, S. , Li, H. , Bodi, Z. , Button, J. , Vespa, L. , Herzog, M. and Fray, R.G. (2008) MTA is an Arabidopsis messenger RNA adenosine methylase and interacts with a homolog of a sex‐specific splicing factor. Plant Cell, 20, 1278–1288. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 The amino acid sequence of dcM/dcdM editor.

Figure S2 The amino acid sequence of dcA/dcdA editor.

Figure S3 Schematic representation of the constructions for subcellular localization in tobacco cells.

Figure S4 Luciferase assay of reporter transcript containing m6A motifs in the 3′UTR in tobacco leaves.

Figure S5 Schematic representation of the constructions for validation of the m6A editors in tobacco cells.

Figure S6 The protein abundance analysis of the editors in tobacco leaf cells.

Figure S7 Genome browser view of m6A peaks and designed gRNAs in candidate target genes for m6A editing.

Figure S8 The phenotype of transgenic overexpression of m6A editors with gRNA targeting SHR.

Figure S9 Root phenotype of Arabidopsis seedlings overexpressing dcM and dcA editors targeting SHR transcript.

Figure S10 The phenotype of transgenic overexpression of demethylation editors with gRNA targeting SHR.

Figure S11 The expression pattern of SHR in different genotypic plants.

Figure S12 The phenotype of transgenic SHR overexpression and knockdown plants.

Figure S13 Differentially expressed genes analysis between Col‐0 and dcM‐SHR.

Figure S14 Peak annotation and m6A distribution analysis with MeRIP‐seq data.

Figure S15 The GO enrichment analysis of the non‐specific genes with up‐ and down‐regulated m6A peaks in dcM‐SHR compared with Col‐0.

Table S1 Primers used in this study.

Table S2 dLshCas13a guide RNA sequences used in this study.

Table S3 Synthesized oligonucleotide sequences used in this study.

PBI-22-1867-s001.pdf (1.1MB, pdf)

Data Availability Statement

The MeRIP sequencing data generated in this study are available in the Gene Expression Omnibus (GEO) as GSE221143. Other data generated during this study are included in this article and the Supporting Information files.

Articles from Plant Biotechnology Journal are provided here courtesy of Society for Experimental Biology (SEB) and the Association of Applied Biologists (AAB) and John Wiley and Sons, Ltd

RESOURCES