CRISPR-based m6A modification and its potential applications in telomerase regulation

Telomerase determines cell lifespan by controlling chromosome stability and cell viability, m6A epigenetic modification plays an important role in the regulation of telomerase activity. Using CRISPR epigenome editing to analyze specific m6A modification sites in telomerase will provide an important tool for analyzing the molecular mechanism of m6A modification regulating telomerase activity. In this review, we clarified the relevant applications of CRISPR system, paid special attention to the regulation of m6A modification in stem cells and cancer cells based on CRISPR system, emphasized the regulation of m6A modification on telomerase activity, pointed out that m6A modification sites regulate telomerase activity, and discussed strategies based on telomerase activity and disease treatment, which are helpful to promote the research of anti-aging and tumor related diseases.


Introduction
The research of telomere and telomerase is of great significance to the aging of organism. Telomerase is an eukaryotic ribonucleoprotein (RNP) composed of RNA-protein complex (Blackburn and Collins, 2011). It extends the 3'end of linear chromosome by synthesizing the telomere repeat TTAGGG to maintain telomere length and chromosome stability . Telomerase activity is closely related to tumorigenesis (Shay, 2016;Trybek et al., 2020), cell proliferation and cell aging (Chakravarti et al., 2021). N6-methyladenosine (m 6 A) RNA modification is an important epigenetic modification mode in post-transcriptional regulation (Schmidt et al., 1975;Roundtree et al., 2017a;Tang et al., 2020), which involves almost all aspects of RNA metabolism and affects various physiological and pathological processes by regulating mRNA cytoplasmic transport, splicing, stability, structure and translation (Dominissini et al., 2012). The composition of telomerase fully shows that it has a close relationship with m 6 A modification. The study of the mechanism of m 6 A modification regulating telomerase activity and maintaining telomere length will promote human anti-aging to provide new ideas.
With the application and improvement of CRISPR (clustered regularly interspaced short palindromic repeats)/Cas system (Jinek et al., 2012;Cong et al., 2013), the modified fusion protein dm 6 ACRISPR system can achieve precise and efficient m 6 A site-specific modification in RNA transcripts (Liu et al., 2019;Wilson et al., 2020), this will help to further explore the mechanism of m 6 A modification. In clinical cancer research, it was found that there was a site mutation in the promoter region of telomerase reverse transcriptase (TERT) gene. Use the base editing function of CRISPR system to reduce the transcription and protein expression of TERT, and induce the aging and proliferation stagnation of cancer cells, which verifies the feasibility of activated TERT promoter mutation as a cancerspecific therapeutic target (Killela et al., 2013;Li et al., 2020). Therefore, the CRISPR system technology, combined with the m 6 A modification of RNA and the regulation of telomerase activity, is used to regulate the aging and proliferation of cells in the body and achieve the treatment of various diseases.
In this review, we reviewed and discussed the latest research progress, and found that the CRISPR system was used to carry out m 6 A site-specific modification of RNA, regulate telomerase activity and affect telomere length by regulating telomerase assembly and other processes, which provided a direction for the study of epigenetic modification to regulate cell aging mechanism, and provided a prospect for the future research on cell proliferation and aging.  CRISPR system and m 6 A modification (A) N6-methyladenosine (m 6 A) regulation mechanism. Writers and erasers strictly regulate the presence of m 6 A on transcripts by targeting the m 6 A motif (DRACH). m 6 A is recognized by readers and starts the steps of regulating mRNA stability and translation. The modification system can be extended to include Cas9 (base editor, writer/eraser fusion) and Cas13 (methylation system). (B) Application of CRISPR/ dCas9-ALKBH5/FTO tool in m 6 A modification. (C) Application of CRISPR/dCas13-Writer Complex tool in m 6 A modification; Application of CRISPR/ dCas13-ALKBH5/FTO tool in m 6 A modification.
Frontiers in Cell and Developmental Biology frontiersin.org 04 and has been widely used in gene editing (Hsu et al., 2014;Manghwar et al., 2019;Zhang et al., 2021). According to the composition of Cas effector proteins, CRISPR system is divided into Class I and Class II (Figure 1). These systems use site-specific guide RNA to guide Cas protein and accurately edit site-specific sequences Yu et al., 2022). At present, the most widely used CRISPR systems are Class II Type II Cas9, Type V Cas12 and Type VI Cas13 (Figure 1).
The CRISPR system is mainly used to modify specific target genes in the genome of organisms. The main editors include DNA cytosine base editor (CBE), adenine base editor (ABE) and primer editor (PE) (Kantor et al., 2020). PE is a multi-functional and high-precision genome editor (Anzalone et al., 2019), which is composed of two parts: the leader editor protein and primer editing guided RNA (pegRNA). Using the CRISPR Cas protein targeted DNA to make nicks and the DNA synthesis ability of reverse transcriptase, the sequence encoded by pegRNA can be accurately and efficiently copied into the targeted DNA sequence to achieve accurate editing, including replacement, insertion and deletion (Anzalone et al., 2019;Kantor et al., 2020;Kim et al., 2020). The advantage of PE is that it does not cause DNA double strand breakage, only cutting one strand of DNA, thereby avoiding potential risks such as chromosome loss and rearrangement caused by double strand DNA breakage. Researchers can further improve the accuracy and specificity of PE by optimizing lead editing proteins, pegRNA, and AAV genomic elements, such as introducing engineered Cas9 mutants, especially eSpCas9 and Sniper Cas9 mutants, into PE (Kim et al., 2020). The PE editing efficiency prediction models DeepPrime, DeepPrime FT, and the off target prediction model DeepPrime Off make the design and screening of pegRNAs more convenient and efficient, providing strong guarantees for the future widespread application of PE systems (Yu et al., 2023). Using PE to repair sickle cell anemia (SCD) mutations in hematopoietic stem cells or progenitor cells of patients, the repaired cells are treated for hereditary blood diseases through transplantation (Everette et al., 2023). The gene editing system developed based on CRISPR technology has brought prospects for the research and treatment of genetic diseases.

CRISPR system and RNA editing
At present, CRISPR/Cas9 and CRISPR/Cas13 systems have become tools for the research and application of DNA and RNA epigenetic modification (Figure 2; Figure 3) (Zhan et al., 2019;Kordyś et al., 2022). Nucleic acid endonuclease deficient Cas9 (dCas9)/Cas13 (dCas13) still has the activity of binding enzyme, which can combine with effector protein to regulate the expression of DNA or RNA, becoming an effective method to study gene function and regulation mechanism Liu et al., 2020).
Working principle of CRISPR/dCas9 system and related tools based on CRISPR/dCas9 system development (gene editing, live cell imaging, base editing, methylation modification, histone modification and transcription regulation). Ac, acetylation; Me, Methylation; Dme, Demethylation.
3.1 RNA editing tools: based on CRISPR/ dCas13 system The modification of CRISPR system for gene editing at the DNA level is irreversible, especially the ethical issues involved in the safety Frontiers in Cell and Developmental Biology frontiersin.org of human germ cell and embryonic cell editing cannot be ignored (Leibowitz et al., 2021;Höijer et al., 2022). p53 gene is a tumor suppressor gene that participates in the regulation of cell growth, differentiation, apoptosis and other processes (Fischer et al., 2016). After Cas9 protein was introduced into cells to realize CRISPR/ Cas9 mediated genome editing, p53 pathway was upregulated and DNA repair level was increased. Cas9 protein induces p53 pathway activation and p53 mediated DNA damage response (Enache et al., 2020). These findings are of great significance for the correct application of CRISPR/Cas9 mediated genome editing (Enache et al., 2020;Sinha et al., 2021). Therefore, the use of CRISPR/ Cas9 technology in human pluripotent stem cells (hPSCs) cell replacement therapy should be carefully carried out and the p53 function of hPSCs cells should be monitored (Ihry et al., 2018). The modification of Cas13 protein at the RNA level successfully avoids irreversible permanent changes to the genome, and is an important tool for studying the most abundant m 6 A modification on RNA. At the same time, it plays an important role in studying the structure and function of telomerase composed of RNA and protein.
In terms of RNA editing, the CRISPR system has been deeply modified and applied to mRNA epigenetic modification research. The m 1 A modification detection method based on the CRISPR/ Cas13a system has been successfully used to identify m 1 A in 28S rRNA . The catalytic inactivation of RfxCas13d (dCasRx) is fused with the m 1 A demethylase ALKBH3, and the dCasRx ALKBH3 fusion protein can mediate effective demethylation of m 1 A modified RNA, known as Reengined m 1 A modification valid eraser ("REVER"), providing a tool for further elucidating the relationship between m 1 A modification of specific transcripts and their phenotypic results (Xie et al., 2021). m 1 A regulates the level of glycolysis in tumor cells by regulating the expression of ATP5D in the mitochondrial ATP synthase F1 domain. The dm 1 ACRISPR system can upregulate the expression of ATP5D through targeted removal of ATP5D m 1 A modification, resulting in an increase in the level of glycolysis of Application of CRISPR/Cas9 system in regulating telomerase and telomere (A) Use CRISPR/Cas9 and CRISPR/dCas9 systems to cut telomeres through telomerase to produce DNA damage and induce cancer cell death. (B) Use CRISPR/Cas9 system to introduce TGS1 gene frameshift mutation to realize the deletion of TGS1 hypermethylation enzyme and promote the increase of telomerase RNA and telomere elongation.
Frontiers in Cell and Developmental Biology frontiersin.org tumor cells (Wu et al., 2022). This is similar to using the CRISPR system to study m 6 A modification, where endogenous editing studies can be conducted by identifying the targets of epigenetic modifications on mRNA such as m 1 A and m 5 C. Because the CRISPR/RfxCas13d (CasRx) related transcriptome epigenetic modification editor has the characteristics of small size and high editing efficiency Zhang et al., 2018), which is suitable for packaging into lentivirus vector for gene function research. At present, CasRx has been successfully used to knock down specific mRNA transcripts in zebrafish embryos (Kushawah et al., 2020), and to mediate RNA targeted treatment of age-related macular degeneration in model mice ).

CRISPR system and m 6 A modification
As an important biological function of RNA modification, m 6 A modification widely exists in almost all types of RNA molecules in cells (Yang et al., 2018;Hu et al., 2022). In the regulation of m 6 A modification, combining the modified protein specific domain with the inactivated CRISPR protein can produce a new precise editing tool for RNA methylation modification ( Figure 4) (Li et al., 2020;Wilson et al., 2020;Kordyś et al., 2022). Liu et al. designed m 6 A modified eraser by combining CRISPR/Cas9 with demethylase ALKBH5 or FTO to realize RNA site-specific demethylation (Liu et al., 2019). Considering the important regulatory role of m 6 A modification on RNA in the nucleus, based on the RNA-targeted endonuclease system CRISPR/Cas13, an editor for targeted RNA methylation (TRM) was constructed, which became a new accurate editing tool for m 6 A modification. The editor can achieve efficient and accurate editing of m 6 A modification of RNA in nucleus and cytoplasm through nuclear export-signal (NES) and nuclear localization signal (NLS) (Wilson et al., 2020). The dm 6 ACRISPR editing tool can realize m 6 A modification of RNA sites, providing a more powerful weapon for indepth research on the function of m 6 A modification (Table 1).

CRISPR system and telomerase
Telomerase, as an enzymatic RNP complex, plays a role of reverse transcriptase in the process of telomere elongation, and is significantly associated with cell aging and tumorigenesis . In cancer cells (Bajaj et al., 2020;Negrini et al., 2020;Wu et al., 2020), hematopoietic stem cells (Celtikci et al., 2021) and germ cells (Dogan and Forsyth, 2021;Lupatov and Yarygin, 2022), telomerase showed high activity (Demanelis et al., 2020). Cancer is closely related to a series of changes in intracellular genome and epigenome (Ushijima et al., 2021). Telomerase is silent in most normal somatic cells, but activated in 90% of cancer cells, making it an excellent target for cancer treatment. In the treatment of cancer, all kinds of telomerase activity inhibitors have failed due to their side effects. Coats plus (CP) is a rare autosomal recessive disease caused by CTC1 mutation, which is important for maintaining telomere length. CTC1L1142H mutation caused telomere damage. The point mutation of CTC1 using CRISPR/ Cas9 technology confirmed that the interaction between CTC1 and STN1 is necessary to inhibit telomerase activity (Gu et al., 2018). Combining the biological functions of CRISPR/Cas9 and telomerase, the development of telomerase activating gene expression (Tage) has gradually become a new cancer gene therapy method. The Tage system consists of three components: the effector gene expression vector carrying 3'telomerase recognition rod end, the dCas9-VP64 expression vector and the sgRNA artificial transcription factor expression vector targeting the telomere repeat sequence. Using AAV as a gene vector, the Tage system can effectively kill cancer cells and safely realize its application in the body . In cancer research using CRISPR system, CRISPR activation screening of targeted gRNA was carried out, gRNA libraries targeting different genes were established, targeted genes in cancer cells were systematically and accurately knocked out, and cancer gene therapy was achieved (Joung et al., 2022;Katti et al., 2022;Ye et al., 2022).
Telomerase activity usually depends on the expression level of TERT, which is the catalytic subunit of RNP complex (Barthel et al., 2017;Wu et al., 2021). The recruitment of telomerase to telomere occurs in the S phase of the cell cycle. By using CRISPR genome editing system and CRISPR-aided nano microscope technology to track telomerase in the nucleus, it is proved that telomerase uses three-dimensional diffusion to search for telomeres, and the recruitment of telomerase to telomere is driven by the dynamic interaction between the rapidly diffusing telomerase protein TERT and telomere protein TPP1 (Schmidt et al., 2016). In the study of human telomerase RNA (hTR) biogenic post-transcriptional modification, the use of CRISPR system consumes trimethyl guanosine synthetase 1 (TGS1). The reduction of trimethylation will increase the coupling of hTR with cap-binding complex (CBC) and Sec1/Munc-18 (Sm) chaperone protein, The accumulation of mature hTR in the nucleus and cytoplasm increases, and the increased hTR is assembled with TERT protein to produce increased active telomerase complex and increased telomerase activity, thus realizing the telomere elongation of cultured human cells. This study provides a new treatment scheme for telomerase dysfunction in telomeric syndrome ( Figure 5) .
In order to further study the activation of telomerase and its activity regulation mechanism, in view of the low editing efficiency of CRISPR/Cas9 at the TERT gene locus, the genome editing method of "pop in/pop out" is used to realize precise modification of
Frontiers in Cell and Developmental Biology frontiersin.org endogenous TERT gene sites in cells. This method provides a powerful tool for studying the biological function of telomerase using CRISPR/Cas9 (Kühn and Chu, 2015;Xi et al., 2015). Thus, the emergence of CRISPR system will provide an important tool for human research on telomerase and the regulation mechanism of cell aging.
6 dm 6 ACRISPR system and telomerase As a repeat DNA sequence at the end of chromosome, telomere shortening is considered as a biological marker of cell aging (Al-Turki and Griffith, 2023). At each cell division, 50-100 pairs of base pairs will be lost in the chromosome end sequence, resulting in cell aging and even death (Blasco, 2005;Rossiello et al., 2022). Telomerase contains specialized TERT and telomerase RNA (TER), and has its own template and catalytic core required by TERT (Cash and Feigon, 2017;Jiang et al., 2018;Wang et al., 2019). In most human cancers, the increase of telomerase level makes cancer cells have the ability to proliferate indefinitely (Roake and Artandi, 2020). According to the characteristics of telomerase structure, composition and epigenetic modification (Figure 6;  Figure 7), the telomere repeat sequence at the end of chromosome is extended to maintain the stability of genome, FIGURE 7 Relationship between telomeres and telomerase (A) Cell proliferation and telomere length reduction. Telomere is a repetitive DNA structure at the top of the chromosome. When the cell division DNA replicates, the telomere will protect the integrity of the chromosome. The activity of telomerase in normal cells was inhibited, and the telomere gradually shortened and disappeared with the continuous cell division. Chromosomes are finally completely exposed, cells cannot proliferate, DNA molecules degrade, and life ends. (B) The life cycle of telomerase and its regulation mechanism. Telomeraseprotein RNA complex uses the non-coding RNA subunit hTR as a template, and the reverse transcriptase TERT catalyzes the telomere elongation. The life cycle of telomerase includes post-transcriptional modification (PTM) and maturation of hTR, intracellular localization, and effective assembly with TERT until the formation of a whole enzyme that can prolong telomeres.
Frontiers in Cell and Developmental Biology frontiersin.org and the gradual loss of telomere caused by genome replication is offset. These are important for studying cell proliferation and delaying cell aging Sekne et al., 2022). The abnormal modification of RNA methylation is closely related to a series of cancer occurrence, and studying the relationship between m 6 A modification and tumor occurrence is of great significance for the treatment of cancer. In liver cancer research, it was found that methyltransferase METTL14 has a dual effect of promoting cancer cell proliferation and differentiation and inhibiting cancer cell metastasis Chen et al., 2018); Overexpression of METTL5 promotes the growth, proliferation, migration, and invasion of liver cancer, knockdown of METTL5 promotes cell apoptosis, and inhibits the growth, proliferation, migration, and invasion of liver cancer . METTL3 has carcinogenic function in human liver cancer, and downregulation of METTL3 can weaken the tumorigenicity and lung metastasis of liver cancer (Chen and Wong, 2020). In glioblastoma, METTL3 can promote the maintenance and

FIGURE 8
Application of m 6 A editing tool of dCas13b-METTL3 in telomerase activity regulation.
Frontiers in Cell and Developmental Biology frontiersin.org radiation resistance of glioblastoma stem cells and inhibit their selfrenewal and proliferation (Cui et al., 2017;Visvanathan et al., 2018). Inhibition of FTO expression can hinder the growth, differentiation and self-renewal of glioblastoma stem cells (Cui et al., 2017). ALKBH5 can promote stem cell self-renewal and proliferation (Zhang et al., 2017). Overexpression of ALKBH5 was found in breast cancer research to enhance the enrichment of breast cancer stem cells (BCSC) (Zhang et al., 2016). In lung cancer and bladder cancer, METTL3 knockout can reduce the growth, survival and invasiveness of lung cancer cells, as well as the proliferation, invasion, in vitro survival and in vivo tumorigenicity of bladder cancer cells (Lin et al., 2016;Han et al., 2019). The m 6 A modification

FIGURE 9
The mechanism of the m 6 A editing tool of CRISPR system to regulate telomerase activity and maintain telomere length in the p53 signal pathway. Frontiers in Cell and Developmental Biology frontiersin.org is closely related to the occurrence of cancer, and the m 6 A editing tool based on the CRISPR system will help to analyze the correlation mechanism between m 6 A modification and cancer occurrence. At present, the dm 6 ACRISPR editing tool is constructed by combining the catalytically inactivated Cas protein with the m 6 A modification related protein (Li et al., 2020). This laid a foundation for studying the relationship between epigenetic modification and telomerase function and exploring the mechanism of m 6 A modification on telomerase activity regulation.
Telomerase structure. Telomerase is a ribonucleoprotein complex, which is composed of scaffold non-coding human telomerase RNA (hTR), telomerase reverse transcriptase (TERT) and related cofactors. Telomerase is composed of two RNA-linked structures. One is the H/ACA domain of hTR, which is composed of two groups of dyskerin complex (dyskerin, NHP2, NOP10 and GAR1) and TCAB1. The other contains the catalytic core, where hTR and TERT surround the telomere substrate. The two are connected through the CR4/5 domain of hTR.

Regulation of telomerase activity by m 6 A modification
RNA epigenetic modifications commonly include 5-methylcytidine (m 5 C) (Bohnsack et al., 2019), N6-methyladenosine (m 6 A) (Oerum et al., 2021), N7-methylguanosine (m 7 G) (Malbec et al., 2019), N1methyladenosine (m 1 A) , inosine (I) (Srinivasan et al., 2021), and pseudo uracil (Ψ) And dihydrouracil (D) (Haruehanroengra et al., 2020). m 6 A modification is closely related to many kinds of carcinogenesis, and altered m 6 A modification is widely involved in the progression of various tumorigenesis (Gu et al., 2020;. Deeply study m 6 A modification by regulating telomerase activity to maintain telomere homeostasis and genome stability is of great significance to clarify the role of m 6 A modification in cell aging and carcinogenesis (Table 2). Through Pan-Cancer Analysis of Whole Genomes (PCAWG) analysis of m 6 A modification of telomerase components, it was found that in most cancers, the expression level of telomerase components was positively correlated with methylase METTL3, negatively correlated with methylase METTL14, negatively correlated with demethylase FTO, negatively correlated with reading proteins YTHDC1, YTHDC2, YTHDF3 and FMR1, and positively correlated with reading proteins HNRNPC, HNRNP2B1, YTHDF1 and RBMX (Wang et al., 2023). These showed that there was a close relationship between telomerase component activity and m 6 A regulatory factors. With the help of the established CRISPR/ dCas13 system to accurately edit the m 6 A modification platform, it is proved that the METTL3-HMBOX1 axis regulates telomere recruitment and telomere length related to telomerase in cancer cells, and leads to DNA damage reaction (Figure 8) (Lee et al., 2021). METTL3 promotes the stabilization of p53 protein and the expression of target genes in response to DNA damage and carcinogenic signals through catalytic activity dependent and independent mechanisms Raj et al., 2022). In addition, METTL3-m 6 A-p53 axis may be a potential target for the treatment of hepatocellular carcinoma (HCC) (Ke et al., 2022). Therefore, we can use CRISPR system to modify specific target genes with m 6 A, and regulate telomerase activity by regulating p53 signal pathway to maintain telomere homeostasis (Figure 9). dCas13b-METTL3, a m 6 A editing tool based on CRISPR system, proves that METTL3-catalyzed HMBOX1 methylation is involved in regulating telomerase recruitment, resulting in telomere loss in cancer cells, and m 6 A is involved in carcinogenesis.

FIGURE 11
Regulation of telomere by m 6 A modification of NLS-dCasRx-NLS-METTL3 system.

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The m 6 A editing tool of CRISPR system modifies mRNA with m 6 A, affects telomerase activity through p53 signal pathway, participates in phosphorylation of PKC and AKT or dephosphorylation of PP2A, telomere shortening leads to DNA damage, and activates p53 signal pathway.
Through its reading protein, m 6 A modification is widely involved in biological processes such as pre-mRNA splicing, RNA output, mRNA translation and RNA degradation, and regulates the stability of targeted mRNA . In the study of m 6 A reading protein, it was found that proteins containing YTH domain (YTHDF1 and YTHDC1) used YTH domain to recognize m 6 A modification, YTHDF1 and YTHDF3 worked together to affect the translation of m 6 A containing mRNA, YTHDF2 accelerated the decay of mRNA, and YTHDC1 affected the nuclear processing of its target, further regulating the function and fate of m 6 A labeled mRNA (Roundtree et al., 2017b;Hsu et al., 2017;Shi et al., 2017).
Knockout of YTHDF1 by CRISPR/Cas9 system will destroy the interaction between YT521-B homologous domain of YTHDF1 and AGO2 (argonaute 2), leading to the transformation of AGO2 droplets into gel/solids deposited in the cytoplasm . In the nucleus, AGO2 interacts with 23 nt sRNA produced by TTS of telomerase RNA component telomerase RNA component (TERC) (position 425-447), which is called terc-sRNA. TERT and TERC constitute the core telomerase that maintains telomere length. As an RNA-binding protein, AGO2 has been found to promote telomerase activity and stimulate the association between TERT and TERC ( Figure 10). AGO2 depletion leads to shorter telomeres and lower cell proliferation rate in vitro and in vivo (Laudadio et al., 2019). By regulating the recognition protein YTHDF1, it can regulate the consumption of AGO2 in the cytoplasm, affect the content of AGO2 in the nucleus, and lead to the change of telomerase activity in cells, which may lay the foundation for new therapeutic targets of tumor and telomeric diseases.
6.2 Site-directed modification of telomerase by dm 6 ACRISPR system After CRISPR/Cas9 system, CRISPR/Cas13 system of type VI belongs to a known type that specifically binds and cleaves exogenous RNA (Abudayyeh et al., 2016;Shmakov et al., 2017;Smargon et al., 2017). CRISPR/Cas13 system can resist pathogenic RNA virus or regulate gene expression, and promote the knockout of mRNA, circular RNA and non-coding RNA (Wessels et al., 2020;Li et al., 2021). In addition, CRISPR/Cas13 system has been used for RNA modification in vivo, including editable regulation of selective splicing, A-to-I and C-to-U editing and m 6 A modification (O'Connell, 2019;Kordyś et al., 2022). Using CRISPR/ Cas13 system, m 6 A can be added to specific RNA sites in a targeted way to achieve precise m 6 A modification at specific RNA sites. Since the methylation and demethylation process of m 6 A mainly occurs in the nucleus, two nuclear localization signal (NLS) peptides are added to dCasRx-METTL3 and dCasRx-ALKBH5 editors to realize the nuclear localization of the editing complex, which are called NLS-dCasRx-NLS-METTL3 and NLS-dCasRx-NLS-ALKBH5 (Xia et al., 2021). m 6 A methyltransferase

FIGURE 12
Regulation of telomerase assembly by m 6 A modification of NLS-dCasRx-NLS-ALKBH5 system m 6 A gene editing tool NLS-dCasRx-NLS-ALKBH5 locates dCasRx-ALKBH5 in the nucleus to achieve specific demethylation. Use the gene editing tool dCasRX-ALKBH5 to modify the m 6 A demethylation of telomerase hTR, regulate the assembly of telomerase components TCAB1 and DKC1, and reduce cell telomerase activity.
Frontiers in Cell and Developmental Biology frontiersin.org METTL3 can increase the methylation modification level of telomerase related gene Cbf5 mRNA, promote its transcription and translation, and enhance telomerase activity (Jiang et al., 2021). As a nuclear protein reverse transcriptase, telomerase is composed of RNA template and catalytic protein . There is a 5-nt GGACU sequence with m 6 A common motif matching in the H/ACA scaRNA structure of hTR , adenosine in the motif (A435) is located in the double stranded region of the RNA, suggesting that its secondary structure may be affected by m 6 A modification (Liu et al., 2015). The double stranded structure of the H/ACA scaRNA domain of hTR has been shown to be important for the assembly of telomerase complexes (Zhang et al., 2011). Overexpression of demethylase ALKBH5 leads to a decrease in the assembly efficiency of TCAB1 and DKC1 on telomerase, resulting in a decrease in telomerase activity. This may be mediated by modifying hTR to regulate telomerase assembly and function . If telomerase activity is regulated by m6A modification, we consider attempting to achieve precise regulation using the nuclear localization CRISPR system combined with dCasRx and NLS. Assuming that the NLS-dCasRx-NLS-METTL3 system overexpressing METTL3 promotes Cbf5 transcription and translation (Figure 11), enhancing telomerase activity, and using NLS-dCasRx-NLS-ALKBH5 overexpressing ALKBH5 to remove m 6 A modification on hTR, Studying the regulation of TCAB1 and DKC1 assembly on telomerase by m 6 A modification ( Figure 12) provides new insights into the potential application of CRISPR based m 6 A modification in telomerase regulation. m 6 A gene editing tool NLS-dCasRx-NLS-METTL3 locates dCasRx-METTL3 in the nucleus to achieve specific methylation. Using the gene editing tool dCasRX-METTL3, the methylation modification level of Cbf5 mRNA was increased, the transcription and translation level of Cbf5 was enhanced, and Cbf5, as a component of telomere synthetase, increased telomere synthetase activity and regulated telomerase activity.
7 Conclusions and future prospects CRISPR gene editing system, as the most revolutionary breakthrough in the field of biotechnology, is an unprecedented tool to cure human genetic diseases (Gillmore et al., 2021;Fox et al., 2022). m 6 A modification plays an important role in almost allimportant biological processes Boulias and Greer, 2022). Telomerase is highly active in stem cells, immune cells and germ cells to maintain telomere length (Jiang et al., 2018;Wan et al., 2021). Using CRISPR system to study the regulation mechanism of m 6 A modification on telomerase activity is of great significance for exploring the mechanism of cell proliferation and aging.
In this review, we systematically describe the latest application of CRISPR system in m 6 A modification and the regulation of telomerase activity, providing ideas for understanding the basic mechanism of regulating cell aging. When considering that m 6 A is the most common, frequent and conservative internal modification, and that telomerase activity is inhibited in normal cells, but remains high in most cancer cells, it is reasonable to propose that further exploring the mechanism of m 6 A modification on telomerase activity regulation will help to identify and develop gene therapy that can fight aging and treat cancer. It is now clear that the expression and activity of these proteins are essential for the correct regulation of the cell's non-stop replication process. Strong evidence has emerged about the various functions of these proteins and the corresponding functions of targeted RNA in stem cells, immune cells, germ cells and sperm. So as we continue to decipher the epigenetic modification of m 6 A and the biology of cell proliferation and aging, we will have an important and indepth understanding of the molecular mechanism of physiological and pathological cell aging.

Author contributions
MY, MW, YX, and ZC drafted the manuscript. YL, ZZ, and HG designed and revised the manuscript. All authors contributed to the article and approved the submitted version.