A crisper-cas protein for epigenetic editing and applications thereof

Abstract

The application provides a CRISPR-Cas protein for epigenetic editing and application thereof, and belongs to the technical field of nucleic acid editing.The application provides a PmCas12m protein for epigenetic editing, and the amino acid sequence is shown as SEQ ID NO:1.Meanwhile, the application obtains a group of PmCas12m protein mutants for epigenetic editing based on structure optimization, and the high-efficiency epigenetic editing capability of the PmCas12m protein is improved.The CRISPR-Cas12m system based on the PmCas12m protein or the mutant thereof can efficiently realize epigenetic regulation of prokaryotes and eukaryotes, and effectively realize the purpose of antiviruses, and provides a new strategy for virus treatment.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid editing technology, specifically relating to a CRISPR-Cas protein for epigenetic editing and its applications. Background Technology

[0002] Epigenetic regulation plays a crucial role in almost all biological processes, and its dysregulation often leads to abnormal gene expression and triggers various diseases. Developing precise and efficient epigenetic regulatory systems has become an important research direction for disease treatment. In recent years, CRISPR / dCas-based epigenetic editing systems have provided a highly efficient editing tool for the field of epigenetic modification research by fusing nuclease-inactivated Cas proteins (dCas) with various epigenetic effector domains to precisely regulate the epigenetics at specific genomic sites, thereby altering gene expression and cell fate. Currently, the most widely used epigenetic editing tools are CRISPR / dCas9 and its mutant systems; however, these systems typically have large molecular weights, which increase further when the effector domains to be fused are added. This significantly limits the application of AAV vector-based in vivo therapy. Therefore, developing a smaller, more functionally efficient dCas protein that can be efficiently delivered via a single AAV vector is key to advancing the clinical translation of epigenetic editing tools.

[0003] In recent years, researchers have discovered three novel CRISPR-Cas systems (Cas12c, Cas12k, and Cas12m) and a class of TnpB-like nuclease-free inhibitory systems (TldRs) while exploring new CRISPR-Cas systems. These systems all possess the characteristic of lacking DNA cleavage activity and exhibit precise targeting capabilities. However, Cas12c's relatively large molecular weight limits its application in AAV-mediated gene delivery, and its editing capabilities in eukaryotic cells remain unknown. Cas12k exhibits low activity in eukaryotic cells, limiting its application in human cells. Furthermore, the function of TldRs in human cells is unclear, further increasing the uncertainty of their practical application. In contrast, Cas12m not only stably binds to double-stranded DNA but also possesses a small molecular weight and abundant prokaryotic homologs, making it a highly promising epigenetic editing tool; however, whether it possesses epigenetic editing capabilities in human cells is also unknown. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a novel PmCas12m protein for epigenetic editing, which has a highly efficient epigenetic editing capability in human cells.

[0005] Another objective of this invention is to provide a mutant of the PmCas12m protein for epigenetic editing, which, through reasonable optimization and molecular weight reduction, yields a mutant xCas12m with a smaller molecular weight and extremely high epigenetic editing efficiency.

[0006] This invention provides a PmCas12m protein for epigenetic editing, the amino acid sequence of which is shown in SEQ ID NO:1.

[0007] This invention provides a PmCas12m protein mutant for epigenetic editing, wherein the PmCas12m protein mutant includes xCas12m or a mutant formed by substituting at least one of the following amino acid residues based on the PmCas12m protein described in the above technical solution: S128Q, A143R, A146C, and E147H;

[0008] The amino acid sequence of xCas12m is shown in SEQ ID NO:2.

[0009] Preferably, the mutant formed by the simultaneous presence of A143R, A146C and E147H mutations on the basis of PmCas12m protein is PmCas12m-RCH.

[0010] The present invention provides a fusion protein, which is formed by fusing the PmCas12m protein or the PmCas12m mutant with an epigenetic effector domain.

[0011] Preferably, the epigenetic effector domain includes at least one of the following: a transcriptional repression domain, a transcriptional activation domain, and a domain regulating DNA methylation.

[0012] Preferably, the transcriptional activation domain includes the VP64-P65AD-Rta effector;

[0013] The transcriptional repression domain includes KRAB-MeCP2 and / or CRISPRoff.

[0014] The present invention provides a CRISPR-Cas12m system for epigenetic editing, comprising sgRNA and at least one of the following: the PmCas12m protein, the PmCas12m mutant, or the fusion protein.

[0015] The present invention provides a vector composition for epigenetic editing, comprising a first recombinant vector containing sgRNA and a second recombinant vector containing a nucleic acid molecule for encoding a protein;

[0016] The protein includes at least one of the PmCas12m protein, the PmCas12m mutant, or the fusion protein.

[0017] Preferably, the first recombinant vector containing sgRNA further includes a spacer fragment;

[0018] The length of the spacer segment is 16 to 20 nt.

[0019] The present invention provides the use of the PmCas12m protein, the PmCas12m mutant, the fusion protein, the CRISPR-Cas12m system, or the vector composition in epigenetic regulation for non-therapeutic purposes.

[0020] Preferably, the epigenetic regulation includes prokaryotic epigenetic regulation and / or eukaryotic epigenetic regulation.

[0021] The present invention provides the use of the PmCas12m protein, the PmCas12m mutant, the fusion protein, the CRISPR-Cas12m system, or the vector composition in the preparation of antiviral drugs.

[0022] Preferably, the virus includes hepatitis B virus.

[0023] This invention provides a PmCas12m protein for epigenetic editing, the amino acid sequence of which is shown in SEQ ID NO:1. Based on MmCas12m (WP_061006603.1), this invention screens homologous proteins using an HMM model, and then obtains the PmCas12m protein through structural prediction and candidate protein verification. The PmCas12m protein possesses flexible 5'-YTN-3' PAM recognition characteristics, binding only to DNA sequences and exhibiting no cleavage activity. PmCas12m inhibits target gene expression by binding to double-stranded DNA and possesses highly efficient epigenetic editing capabilities in human cells, which can be used to resist the invasion of foreign bacteriophages or viruses. Further three-dimensional structural analysis using cryo-electron microscopy revealed that the PmCas12m protein exerts its epigenetic editing capabilities by forming a PmCas12m-crRNA-target gene DNA complex. The PmCas12m protein provided by this invention has strong epigenetic editing capabilities, enriches the current Cas12m protein library, and can be used for epigenetic regulation and clinical gene therapy.

[0024] This invention provides a PmCas12m protein mutant for epigenetic editing, comprising xCas12m or a mutant formed by substituting at least one of the following amino acid residues into the PmCas12m protein: S128Q, A143R, A146C, and E147H; the amino acid sequence of xCas12m is shown in SEQ ID NO:2. This invention, through saturation mutation screening of 137 amino acid residues, obtained at least one mutation at one of the four mutation sites S128Q, A143R, A146C, and E147H, which exhibits improved epigenetic editing efficiency. This invention further performs C-terminal and N-terminal deletions on the amino acid sequence to obtain xCas12m, which not only maintains its high epigenetic editing efficiency but also further reduces the protein molecular weight, thus improving protein delivery efficiency and promoting the development of the field of efficient epigenetic editing. Attached Figure Description

[0025] Figure 1 The results of PAM screening for PmCas12m protein are shown below. a) Schematic diagram of the PmCas12m protein screening process, where red asterisks indicate conserved DED residues in the RuvC domain; b) Schematic diagram of the PAM-SCANR in vivo PAM screening platform identifying PAM CRISPR sequences; c) GFP-positive assay results (left) and statistical bar chart (right) of PmCas12m-activated E. coli cells containing functional PAM; d) Statistical results of PpCas12m and MkCas12m-activated GFP-positive E. coli cells containing functional PAM; e) Statistical graph of the recognition motifs of PmCas12m, PpCas12m, and MkCas12m; f) Results of small RNA assays enriched in the minimal CRISPR-PmCas12m phylogenetic sequence; Data represent the mean ± standard deviation of three biological replicates; ***P < 0.001.

[0026] Figure 2The results of PmCas12m-mediated gene expression silencing assays in *E. coli* cells are shown in Figure a. A schematic diagram of the two-color fluorescence interference detection, where arrows indicate sgRNA targets designed for PmCas12m; Figure b. Comparison of RFP and GFP fluorescence levels between cells expressing the targeted sgRNA and those not expressing the targeted sgRNA (top figure), and comparison of RFP and GFP mRNA levels between cells expressing the targeted sgRNA and those not expressing the targeted sgRNA (bottom figure); Figure c. Evaluation of transformation efficiency and RFP and GFP expression in *E. coli* cells using PmCas12m fluorescence interference detection; Figures de. The results of PmCas12m-mediated gene expression silencing assays are shown, where arrows indicate the sgRNA targets used. NES indicates non-essential sequences; data are the mean ± standard deviation of three biological replicates; ***P < 0.001;

[0027] Figure 3 This is a schematic diagram of the nine sets of second carrier structures designed for this invention;

[0028] Figure 4 The results of PmCas12m-mediated epigenome editing activation of reporter genes in human cells are shown in the following figures: a) Design of the PmCas12m-VPR activation system in TRE3G-GFP HEK293T cells (top figure) and flow cytometry measurement of PmCas12m-VPR activation efficiency for GFP (right figure); b) GFP activation efficiency of different PmCas12m-VPR plasmid designs (#1-#9); c) GFP activation efficiency of PmCas12m-VPR mediated by different spacers; d) Effect of mispairing of 1-nt or 2-nt spacers on GFP activation efficiency of PmCas12m-VPR. Data represent the mean ± standard deviation of three biological replicates.

[0029] Figure 5 The structure determination results of the PmCas12m-crRNA-DNA complex are shown in Figure a. The structural domain of PmCas12m is shown in Figure b. The schematic diagram of crRNA and DNA is shown in Figure c. The recognition sites of crRNA and DNA are shown in Figure c.

[0030] Figure 6The results are based on optimization guided by the PmCas12m structure. a) is a schematic diagram of the PmCas12m-crRNA-DNA complex structure; b) shows the DMS method evaluation of TRE3G-GFP. Schematic diagram of GFP activation efficiency in HEK293T cells; c shows the effect of a single mutation on GFP activation efficiency, with the upper figure showing the PAM of the TTC motif and the lower figure showing the PAM of the TTA motif. Colors indicate mutation effects, with red indicating more efficient mutations; d is a heatmap of key amino acid substitutions in the PAMs of the TTC (left) and TTA (right) motifs; e shows the activation efficiency of the PmCas12m-VPR variant for GFP measured by flow cytometry, using PAMs of the TTC (left) and TTA (right) motifs; f is a schematic diagram of rationally designed PmCas12m reduction based on structural analysis; g shows the effect of the PmCas12m deletion variant using the TTC (left) and TTA (right) PAMs on GFP activation activity; data are expressed as mean ± standard deviation of three biological replicates, ***P < 0.001, **P < 0.01, *P < 0.05, ns indicates no significant difference;

[0031] Figure 7 The results of xCas12m-mediated epigenetic editing for endogenous gene regulation in human cells are shown in Figure a. Figure a is a schematic diagram of the xCas12m-VPR activation system; Figures b and h compare the activation efficiencies of xCas12m-VPR, PmCas12m-VPR, and controls in activating HBG1 / 2(b), CD34(c), IL2RA(d), FOXA3(e), IL1RN(f), ASCL1(g), and TTN(h); Figure i is a schematic diagram of the xCas12m-KRAB-MeCP2 inhibition system; Figure j and p compare the inhibition efficiencies of xCas12m-KRAB-MeCP2, PmCas12m-KRAB-MeCP2, and controls in inhibiting BRCA1(j), CHEK1(k), CXCR4(l), MET(m), RBM3(n), HINT1(o), and B2M(p); Data represent the mean ± standard deviation of three biological replicates.

[0032] Figure 8The results of applying xCas12m-CRISPRoff-mediated epigenetic editing to HBV treatment are shown in the following figures: a) Schematic diagram of the xCas12m-CRISPRoff inhibitory system construct; b) Circular (left) and linear (right) diagrams of the HBV genome, where XP represents the X promoter; CP represents the core promoter; SPI represents the PreS1 promoter; SPII represents the PreS2 promoter; EN I represents enhancer I; EN II represents enhancer II; c) Measurement results of HBeAg (left) and HBsAg (right) levels in the supernatant of HBV-infected HepG2-NTCP cells after transfection with the xCas12m-CRISPRoff system; d) HBV total RNA (left) and pgRNA (right) levels in HBV-infected HepG2-NTCP cells after transfection with the xCas12m-CRISPRoff system; e) HBV total RNA levels in HepG2 cells (left) and HepAD38 cells (right) after transfection with the xCas12m-CRISPRoff system; f) Transfection... pgRNA levels in HepG2 cells (left) and HepAD38 cells (right) after transfection with the xCas12m-CRISPRoff system; g represents RNA-seq analysis results showing changes in the HBV transcriptome in HepG2-NTCP cells infected with HBV under xCas12m-CRISPRoff-mediated inhibition of key viral elements using two representative sgRNAs; independent experimental replicates are represented as rep1 and rep2, respectively; h represents total HBV in HepG2-NTCP cells transfected with the xCas12m-CRISPRoff system. DNA levels (in culture supernatant); i represents the levels of total HBV DNA (left) and cccDNA (right) in HBV-infected HepG2-NTCP cells after transfection with the xCas12m-CRISPRoff system; j represents a schematic diagram of the xCas12m-CRISPRoff inhibitory system based on the AAV vector; k represents a schematic diagram of the in vivo experimental design; ln represents the levels of HBeAg (l), HBsAg (m), and circulating HBV DNA (n) in mouse serum at specified time points; data represent the mean ± standard deviation of three biological replicates; ***P < 0.001, **P < 0.01, *P < 0.05, ns indicates no significant difference. Detailed Implementation

[0033] This invention provides a PmCas12m protein for epigenetic editing, the amino acid sequence of which is shown in SEQ ID NO:1 (MKNIAVQTNKAATESAVTVFRYGLLAPINWGRDVEDELYRMNALWNKLVEIERANRERY ).

[0034] In this invention, 15 candidate proteins with high structural similarity to MmCas12m (WP_061006603.1) were screened through bioinformatics analysis. The activity of the candidate proteins and the identification of PAM and crRNA were verified using a PAM-SCANR experiment. The results showed that mCas12m, MkCas12m, and PpCas12m exhibited strong activities, with PpCas12m showing the strongest activity. PAM identification results indicated that PpCas12m recognizes PAMs with the 5'-YTN-3' (Y = C or T, N = A, T, C, and G) motif. In this embodiment, the PAM can be TTA, TTT, CTT, or TTC. Small RNA sequencing results showed that the sgRNA of PpCas12m consists of a 33nt crRNA and a 17nt spacer sequence. PpCas12m only possesses the ability to bind DNA sequences and does not have enzymatic cleavage activity.

[0035] The present invention provides a PmCas12m protein mutant for epigenetic editing, which is formed by the deletion and / or substitution of amino acid residues on the basis of PmCas12m protein, including xCas12m or mutants formed by at least one of the following amino acid residue substitution mutations on the basis of PmCas12m protein: S128Q, A143R, A146C and E147H.

[0036] In one embodiment of the present invention, the PmCas12m protein mutant preferably includes a mutant formed by substituting one, two, three, or four amino acid residues from S128Q, A143R, A146C, and E147H into the PmCas12m protein. When a mutation occurs in the PmCas12m protein mutant, the resulting mutant includes PmCas12m-S128Q(,SEQ ID NO:4)、PmCas12m-A143R(MKNIAVQTNKAATESAVTVFRYGLLAPINWGRDVEDELYRMNALWNKLVEIERANRERYREIISTSPALSEVSERIEALHREREDLIAERKRRRASARSKSKADTADLDERIKAIKAELAPLYEQRKSLAAEAREQQKPLLDRLEAERREAVKAARQSSGCFWPNYNAVIASYEIARKRAMKTGADLRFRRFSREGRLVNQIQGGMSVEDLFSCRHSQVGIRLGGQSRGRQTGTLYVTAYTGRDESGRRIRRNVEFPIILHRPFPKDAVIKEVAVNIRRRSPSVVSGQTETDDGRIIEYGEAEYSVAFTCQTPAPEKSAGSSAAGINIGWKRVSGGLRVATAAFHDGTFEHLILPDEWVKKYERVQALRSGIDDADNEMHAALRQALQGMPLWERDGPMVEGLSDSDHRLLSAIKRAPRAPGRAMDALAWRLKETPNMPFVADLGATIEAWRKARKRMILEMDNLRGKLLARRKDLYRTFAARIAAYAGAIAIDDTDYRQAALVERTDGEDLELHEQARRQRVMAAPYELRLAIEQAAAKRGGYVERHRGSVNHCRACRSRNVSGDIARHCHACGAVFDVDENAALNLLHTLIAGPARAVE,SEQ ID NO:5)、PmCas12m-A146C(MKNIAVQTNKAATESAVTVFRYGLLAPINWGRDVEDELYRMNALWNKLVEIERANRERYREIISTSPALSEVSERIEALHREREDLIAERKRRRASARSKSKADTADLDERIKAIKAELAPLYEQRKSLAAEAREQQKPLLDALECERREAVKAARQSSGCFWPNYNAVIASYEIARKRAMKTGADLRFRRFSREGRLVNQIQGGMSVEDLFSCRHSQVGIRLGGQSRGRQTGTLYVTAYTGRDESGRRIRRNVEFPIILHRPFPKDAVIKEVAVNIRRRSPSVVSGQTETDDGRIIEYGEAEYSVAFTCQTPAPEKSAGSSAAGINIGWKRVSGGLRVATAAFHDGTFEHLILPDEWVKKYERVQALRSGIDDADNEMHAALRQALQGMPLWERDGPMVEGLSDSDHRLLSAIKRAPRAPGRAMDALAWRLKETPNMPFVADLGATIEAWRKARKRMILEMDNLRGKLLARRKDLYRTFAARIAAYAGAIAIDDTDYRQAALVERTDGEDLELHEQARRQRVMAAPYELRLAIEQAAAKRGGYVERHRGSVNHCRACRSRNVSGDIARHCHACGAVFDVDENAALNLLHTLIAGPARAVE,SEQ ID NO:6) and PmCas12m-E147H (SEQ ID NO:7). When two, three, or four mutations are simultaneously performed, the PmCas12m protein mutants preferably include simultaneous mutations of S128Q and A143R, simultaneous mutations of S128Q and A146C, simultaneous mutations of S128Q and E147H, simultaneous mutations of A143R and A146C, simultaneous mutations of A143R and E147H, simultaneous mutations of A146C and E147H, simultaneous mutations of S128Q, A143R and A146C, simultaneous mutations of S128Q, A143R and E147H, simultaneous mutations of A143R, A146C and E147H, and simultaneous mutations of S128Q, A143R, A146C and E147H. When PAM is a TTC or TTA motif, the mutant formed by simultaneous mutations of A143R, A146C, and E147H is named PmCas12m-RCH, which exhibits high epigenetic editing activity. The preferred amino acid sequence of PmCas12m-RCH is as shown in SEQ ID NO:3.

[0037]

[0038] In this invention, the PmCas12m protein mutant also includes mutants obtained by further deletion mutations on the PmCas12m protein or the mutant itself, such as xCas12m. xCas12m is formed by further deleting amino acid sequences 2-13 and 594-601 from PmCas12m-RCH, and is a compact protein. The amino acid sequence of xCas12m is shown in SEQ ID NO:2(), with a length of 581 amino acids. xCas12m retains the epigenetic editing activity of PmCas12m-RCH while further reducing the molecular weight of the protein, thereby improving its cellular delivery efficiency. The results of the embodiments of this invention show that inappropriate deletion mutations can affect the editing activity of the mutant. For example, when further deleting the WED (Δ285-299), RuvC (Δ382-450), or TNB (Δ551-578) regions from xCas12m, the epigenetic editing activity of the resulting mutant is significantly reduced or even completely lost.

[0039] The present invention provides a fusion protein, which is formed by fusing the PmCas12m protein or the PmCas12m mutant with an epigenetic effector domain.

[0040] In this invention, in order to achieve precise epigenetic regulation, the PmCas12m protein or the PmCas12m mutant is expressed by fusing with an epigenetic effector domain. The resulting fusion protein binds to sgRNA to achieve the regulatory purpose of activating and inhibiting the expression of the target gene.

[0041] In this invention, the epigenetic effector domain preferably includes at least one of the following: a transcriptional repression domain, a transcriptional activation domain, and a domain regulating DNA methylation. The transcriptional activation domain preferably includes the VP64-P65AD-Rta effector. The transcriptional repression domain preferably includes KRAB-MeCP2 and / or CRISPRoff. In one embodiment of this invention, to evaluate the epigenomic activation and repression efficiency of xCas12m, PmCas12m protein and xCas12m were fused with either the VP64-P65AD-Rta effector or the VP64-P65AD-Rta effector, respectively, while dCas9, dSpRY, dCasMINI, MmCas12m, denAsCas12f1, and dSpCas12f1 were used as controls. The results showed that both PmCas12m-VPR and xCas12m-VPR could efficiently and stably activate these endogenous genes, and the activation efficiency of xCas12m-VPR was consistently higher than that of wild-type genes. The PmCas12m-VPR model was successfully implemented. Simultaneously, PmCas12m-KRAB-MeCP2 and xCas12m-KRAB-MeCP2 exhibited varying degrees of gene repression in all seven endogenous genes, with superior repression effects compared to dCasMINI, MmCas12m, denAsCas12f1, and dSpCas12f1. In some cases, the repression efficiency of xCas12m-KRAB-MeCP2 was even higher than that of dCas9-KRAB-MeCP2, suggesting that xCas12m-KRAB-MeCP2 may be an effective alternative to the CRISPR-mediated gene repression strategy. These results indicate that PmCas12m and xCas12m possess the potential to be efficient and flexible epigenome editing platforms, laying the foundation for epigenetic regulation research and therapeutic gene regulation applications.

[0042] The present invention provides a CRISPR-Cas12m system for epigenetic editing, comprising sgRNA and at least one of the following: the PmCas12m protein, the PmCas12m mutant, or the fusion protein.

[0043] In this invention, in the CRISPR-Cas12m system, the core function of the sgRNA (single-guide RNA) is to guide the Cas12m protein to accurately recognize and bind to the target gene.

[0044] In this invention, when inhibiting the transcriptional expression of GFP and RFP genes in *E. coli*, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:8 to SEQ ID NO:15. When activating GFP gene expression in eukaryotic cells, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:16 to SEQ ID NO:18. When detecting the epigenomic activation efficiency of xCas12m-VPR, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:19 to SEQ ID NO:25. When detecting the inhibition efficiency of xCas12m-KRAB-MeCP2, the nucleotide sequence of the sgRNA is shown in SEQ ID NO:26 to SEQ ID NO:34.

[0045] The present invention provides a vector composition for epigenetic editing, comprising a first recombinant vector containing sgRNA and a second recombinant vector containing a nucleic acid molecule for encoding a protein; wherein the protein preferably comprises at least one of the PmCas12m protein, the PmCas12m mutant, or the fusion protein.

[0046] This invention does not impose any particular limitation on the construction method of the first or second recombinant vector; any recombinant vector construction method well known in the art can be used. In this embodiment, the backbone vector of the first recombinant vector is preferably an adeno-associated virus (AAV) vector. The first recombinant vector containing sgRNA further includes a spacer fragment; the length of the spacer fragment is preferably 16–20 nt, and more preferably 17 nt.

[0047] The present invention provides the application of the PmCas12m protein, the PmCas12m mutant, the fusion protein, the CRISPR-Cas12m system, or the vector composition in epigenetic regulation.

[0048] In this invention, the epigenetic regulation preferably includes prokaryotic epigenetic regulation and / or eukaryotic epigenetic regulation. The prokaryotes preferably include *Escherichia coli*. The eukaryotes include primates, particularly humans.

[0049] In this invention, the epigenetic regulation method preferably involves fusing the PmCas12m protein or the PmCas12m mutant with an epigenetic effector domain in the target object for expression. Under the action of sgRNA targeting the target gene, a ternary complex of PmCas12m protein (PmCas12m mutant)-sgRNA-target gene is formed. Under the regulation of the epigenetic effector domain, transcriptional activation, transcriptional repression, methylation, or histidine modification of the target gene are achieved, thereby realizing epigenetic regulation.

[0050] The present invention provides the use of the PmCas12m protein, the PmCas12m mutant, the fusion protein, the CRISPR-Cas12m system, or the vector composition in the preparation of antiviral drugs.

[0051] In this invention, the virus in the antiviral agent preferably includes hepatitis B virus (HBV). The antiviral agent preferably inhibits viral antigen expression and viral DNA replication. When used against hepatitis B virus, the nucleotide sequence of the sgRNA in the CRISPR-Cas12m system is as shown in SEQ ID NO:26 or SEQ ID NO:34.

[0052] In another embodiment of the present invention, the xCas12m-CRISPRoff system and its sgRNA were constructed into the same AAV vector, and the resulting recombinant vector was injected into a mouse model infected with HBV. The results showed that xCas12m-CRISPRoff significantly reduced serum HBeAg levels, HBsAg levels, and circulating HBV DNA levels, and could continuously reduce viral products after a single dose. The present invention also verified that xCas12m-CRISPRoff exhibited significant antiviral activity in multiple HBV cell models (HepG2 cells, HepAD38 cells, and HepG2-NTCP cells), effectively inhibiting viral transcription and replication and reducing viral DNA load by targeting key HBV transcriptional regulatory elements. These results indicate that xCas12m-CRISPRoff is a highly efficient and safe epigenetic editing tool, providing a new strategy for HBV treatment.

[0053] The following detailed description, in conjunction with embodiments, of a CRISPR-Cas12m system for epigenetic editing provided by the present invention and its applications, should not be construed as limiting the scope of protection of the present invention.

[0054] Example 1

[0055] Protein structure-guided screening and identification of PmCas12m proteins

[0056] 1. Screening Method

[0057] A. Using the MmCas12m sequence (WP_061006603.1) as the query sequence, two rounds of PSI-BLAST searches were performed in the NCBI non-redundant (NR) database. To reduce the inclusion of irrelevant proteins, strict filtering parameters were set, including an expected value (E value) threshold of 1e-6, a PSI-BLAST threshold of 1e-7, and a query coverage of over 80%. The selected protein sequences were further filtered according to length (400–800 amino acids) and clustered using MMSeq2 with 70% sequence similarity and 70% sequence coverage. Representative sequences from each cluster were aligned using MAFFT-einsi, and the alignment results were divided into three domains: REC, WED, and RuvC. Hidden Markov Models (HMMs) were then constructed for each domain.

[0058] To rapidly and accurately identify CRISPR-Cas12m homologs, potential active CRISPR / Cas candidate systems from 21 bacterial families (a total of 5,663 bacterial genomes) were collected from the NCBI RefSeq database. For full-length proteins, only those matching two or more HMM models were retained to reduce false positives. Subsequently, CRISPR arrays within a 5,000 bp range upstream and downstream of the Cas12m site were detected using CRISPRCasFinder, and those containing only three or more direct repeat sequences (DRs) were considered valid. Finally, 30 CRISPR-Cas12m proteins containing non-DED (Asp-Glu-Asp) motifs were screened, and their structures were predicted using the AlphaFold 3 database. Subsequently, DALI was used for structural alignment with MmCas12m, and the 15 candidate proteins most structurally similar to MmCas12m were selected for functional testing.

[0059] B. 100 ng of PAM library plasmid (containing the GFP coding sequence, purchased from Nanjing Genscript Biotech Co., Ltd.) and 200 ng of CRISPR / Cas12m plasmid (constructed into the pBad33 vector using homologous recombination to extract the candidate protein coding sequence and CRISPR sequence) were electrotransformed into E. coli BW25113 strain lacking the lacI and lacZ genes and the IE-type CRISPR-Cas system. The transformation mixture was inoculated at a 1:100 volume ratio into 5 mL LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and cultured overnight at 37°C. The next day, the culture was diluted 1:100 with PBS, and GFP-positive and GFP-negative cells were sorted by flow cytometry (FACS). Approximately 100,000 single cells were sorted from each experiment and inoculated into 5 mL LB medium containing the corresponding antibiotics, and cultured overnight at 37°C. On day three, GFP-positive and GFP-negative samples were diluted 1:100 with PBS, and GFP-positive and GFP-negative cells were sorted again using FACS, with approximately 100,000 single cells in each category. The sorted cells were then seeded into 5 mL LB medium containing the corresponding antibiotics and cultured overnight at 37°C. Plasmids were then extracted using the EndoFree Mini Plasmid Kit (TIANGEN), and the region approximately 200 bp surrounding the target site was amplified using TransGen's FastPfu Fly PCR SuperMix to add Illumina adapters and sample barcodes. The constructed libraries were sequenced using the Illumina HiSeq platform (PE150). The abundance of each PAM was normalized according to the total number of reads in the sample.

[0060] C. Electroporate 100 ng of PAM library plasmid and 200 ng of CRISPR / Cas12m plasmid into E. coli BW25113 strain lacking the lacI and lacZ genes and the IE-type CRISPR-Cas system. The transformation mixture was inoculated at a 1:100 volume ratio into 5 mL LB medium containing 50 μg / mL kanamycin and 34 μg / mL chloramphenicol, and cultured overnight at 37°C. The next day, the culture was diluted 1:100 with PBS, and the percentage of GFP-positive cells was detected by flow cytometry (FACS).

[0061] D.6-FAM-labeled double-stranded DNA (dsDNA) binding assay: 100 nM PmCas12m and 250 nM sgRNA were incubated in 1× reaction buffer (NEB, Buffer 2.1) at 37°C for 30 min. Then, 20 nM dsDNA was added, and incubation continued at 37°C for another 30 min. The reaction was terminated by adding 2× stop buffer. The reaction products were separated by electrophoresis on a 6% DNA retardation gel at 120 V for 40–60 min.

[0062] E. To verify whether PmCas12m lacks in vitro DNA cleavage activity, a ternary complex was formed according to the above method. The reaction system was then treated with RNase A (NEB) at 37°C for 15 minutes, followed by incubation with Proteinase K (NEB) at 37°C for 15 minutes. The reaction was terminated by adding 2× termination buffer and heated to 95°C for 5 minutes, followed by analysis by 15% TBE-urea PAGE gel electrophoresis.

[0063] F. 100 ng of PAM library plasmid and 200 ng of CRISPR / Cas12m plasmid were electroporated into E. coli strain BW25113 lacking the lacI and lacZ genes and the IE-type CRISPR-Cas system. Total RNA was extracted from E. coli carrying the CRISPR / Cas12m system using the TransGen TransZol Up RNA Extraction Kit. Approximately 60 μg of the extracted RNA was treated with 2 U DNase I (NEB) at 37°C for 30 min to remove DNA contamination, followed by purification by phenol / chloroform extraction. The DNA-free RNA was incubated with 20 U T4 polynucleotide kinase (T4 PNK, NEB) at 37°C for 6 h. Then, 1 mM adenosine triphosphate (ATP; NEB) was added, and incubation continued at 37°C for 1 h. Phenol / chloroform extraction was then performed again for purification. Next, the RNA was incubated with 5 U of RppH (NEB) at 37°C for 1 hour, followed by a final phenol / chloroform extraction purification. 5 μg of the pretreated RNA was used to construct a small RNA library using the VAHTS Small RNA Library Prep Kit for Illumina (Vazyme), following the manufacturer's instructions. The constructed small RNA library was sequenced using the Illumina HiSeq platform (PE150). Sequencing data were analyzed using Bowtie2 and SAMtools.

[0064] See results Figure 1To explore the potential activity of the screened Cas12m candidate proteins, a PAM-SCANR assay was used to determine whether the Cas12m candidate proteins possessed activity and to identify their PAM and crRNA (PAM and crRNA). Figure 1In b). The results show that PmCas12m, MkCas12m (MIRTYKYSLKAPENFAEDCEDELRRMNDLWNRLIEIDRQRERSFKDLCRSTSAEYAAAQDEIEALREPIDNLYDAIRAERIATRSKEPSDELRARRDELLGRRKALWEICKAIQKAIPKESQAPINEVYKTNVKLARQQSGCFWGNYNAVIESFETAKSKAIKDGGRLHFKSFDGSGRFVNQIQGGMTVTELLAGSHSQAQLTNLVTTNKTKGRFAFTAFTGKDDAGKRFRRQLFSEINYHRPIPADGVIKAVEVVKVPHDGKQKYKWHACFTVALPEVDIKHPKRNIAGVNLGWRQFGGRLRVAVVVDDAGKKTEYFVPAELVSKFEAAETIQKAADDARNEMLSWLRTFYQDNRDEAPQEWRESIQGLLRNRPSVDAANHLMTIWRECVFAQEESRRYAAWLKSDAALRRSYTGCRQNAVKWREEIYRHIAKELAERYAVLAVTDTPLSTMSRTKAKDDLAVDNALPESARRNRVIAAIYSLKEWIGKQAAKTGSTVETITGKMTATCHKCGYVAEKRLRGSQYATCKSCGSELELDENAAINCRNHASGAVLISDKPEKTGRFQRAKMAENDFARKIGDNASPLVT,SEQ ID NO:35) and PpCas12m (SEQ ID NO:36) induced strong fluorescence signals, with PmCas12m exhibiting the strongest activity. Figure 1 medium cd).

[0065] PAM identification results showed that PmCas12m recognizes PAMs with the 5'-YTN-3' (Y = C or T) motif, MkCas12m recognizes 5'-YTTS-3' (Y = C or T, S = G or C) PAMs, while PpCas12m recognizes 5'-TTV-3' (V ≠ T) PAMs. Figure 1(e). Given that PmCas12m exhibits the strongest binding activity and more flexible PAM recognition characteristics, it was chosen for subsequent functional studies. To elucidate the sgRNA sequence of PmCas12m, small RNA sequencing was performed, revealing that its sgRNA consists of a 33nt crRNA and a 17nt spacer sequence. Figure 1 (f)

[0066] The above results demonstrate that PmCas12m has the characteristics of binding to double-stranded DNA but not cleaving it, and also has flexible 5'-YTN-3PAM recognition properties, laying the foundation for its application in the fields of epigenetic regulation and gene silencing.

[0067] Example 2

[0068] In Escherichia coli, PmCas12m inhibits the expression of target genes by binding to double-stranded DNA.

[0069] To verify that PmCas12m only has the ability to bind DNA and not to cleave it, a bicistronic expression vector was used, i.e., one promoter simultaneously drives GFP (encoding protein sequence MSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTFAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITHGMDELYK, SEQ ID The expression of NO:37) and the RFP gene (encoding protein sequence MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMRLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHSTGA, SEQ ID NO:38) was used to conduct experiments. Figure 2 (a)

[0070]

[0071] RFP sg1: ACGGTCACGAGTTCGAAATC (SEQ ID NO:8);

[0072] RFP sg2: GGGACATCCTGTCCCCGCAG (SEQ ID NO:9);

[0073] RFP sg3:GAAACCTTCCGGGAAGGACA(SEQ ID NO:10);

[0074] RFP sg4: AGCCATGTAGGTGGTTTTAA (SEQ ID NO: 11);

[0075] GFP sg5: CTGTCAGTGGAGAGGGTGAA (SEQ ID NO: 12);

[0076] GFP sg6: CAGTAGTGCAAATAAATTTA (SEQ ID NO: 13);

[0077] GFP sg7: TAAAGAAGATGGAAACATTC (SEQ ID NO: 14);

[0078] GFP sg8: TGTTGATAATGGTCTGCTAG (SEQ ID NO: 15).

[0079] Plasmid Construction Method: pCas12m and pCRISPR plasmids were constructed using homologous recombination. The plasmids carrying both pCas12m and pCRISPR, along with a bicistronic expression plasmid, were co-transformed into E. coli competent cells. The transformed mixture was then revived (usually at 37°C for about 1 hour) and inoculated into 3 mL LB broth containing the appropriate antibiotics, and cultured at 37°C for 16–20 hours. The expression levels of GFP and RFP were subsequently detected by qRT-PCR. The qRT-PCR primers were:

[0080] RFP-F:GACGGTGCTCTGAAAGGT (SEQ ID NO:39);

[0081] RFP-R: TCGTTGTGGGAGGTGATG (SEQ ID NO: 40);

[0082] GFP-F: ACTTTCGCGTATGGTCTT (SEQ ID NO: 41);

[0083] GFP-R: TAGTTCCCGTCATCTTTG (SEQ ID NO: 42).

[0084] The results showed a decrease in the expression levels of GFP and RFP. Figure 2 (b) The amplification capacity of E. coli was not affected. Figure 2 (c). This indicates that when sgRNA targets different locations in the GFP and RFP sequences, PmCas12m can significantly inhibit the transcription and translation of GFP and RFP, but does not affect the proliferation activity of *E. coli*. However, when PmCas12m targets key regions of the plasmid (e.g., resistance genes or origin of replication), it can induce cell death in *E. coli*. Figure 2 These results indicate that PmCas12m can exert transcriptional repression by binding to DNA, thereby providing adaptive immunity to prevent invasion by foreign bacteriophages or viruses.

[0085] Example 3

[0086] PmCas12m can be used as a validation experiment for an epigenetic editing tool in eukaryotic cells.

[0087] 1. The sgRNA (different sgRNAs were designed for different PAMs, with the corresponding DNA sequences as follows: TTA:CTCCCTATCAGTGATAGAGA, SEQ ID NO:16; TTC:TCTATCACTGATAGGGAGTA, SEQ ID NO:17; TTT:ACTCCCTATCAGTGATAGAG, SEQ ID NO:18) was cloned into pSLQ9834 (Addgene#176272) to construct the first recombinant vector (see...). Figure 3in a). The VP64-P65AD-Rta(VPR) effector (SEQ ID NO: 43, DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSGGSGSQYLPDTDDRHRIEEKRKRTYETFKSIMKKSPFSGPTDPRPPPRRIAVPSRSSASVPKPAPQPYPFTSSLST INYDEFPTMVFPSGQISQASALAPAPPQVLPQAPAPAPAPAMVSALAQAPAPVPVLAPGPPQAVAPPAPKPTQAGEGTLSEALLQLQFDDEDLGALLGNSTDPAVFTDLASVDNSEFQQLLNQGIPVAPHTTEPM LMEYPEAITRLVTGAQRPPDPAPAPLGAPGLPNGLLSGDEDFSSIADMDFSALLSQISSGSGSGSRDSREGMFLPKPEAGSAISDVFEGREVCQPKRIRPFHPPGSPWANRPLPASLAPTPTGPVHEPVGSLTPAPVPQPLDPAPAVTPEASHLLEDPDEETSQAVKALREMADTVIPQKEEAAICGQMDLSHPPPRGHLDELTTTLESMTEDLNLDSPLTPELNEILDTFLNDECLLHAMHISTGLSIFDTSLF) was fused with PmCas12m (SEQ ID NO:1) to form the fusion protein PmCas12m-VPR. The encoding gene of PmCas12m-VPR was cloned into pSLQ9926 (Addgene#176269) to construct the second recombinant vector. Nine versions of the second recombinant vector were constructed based on the positions of three nuclear localization sequences: SV40 NLS (SEQ ID NO: 117, PKKKRKV), c-Myc NLS (SEQ ID NO: 118, PAAKRVKLD), and Nucleoplasmin NLS (SEQ ID NO: 119, KRPAATKKAGQAKKKK). See the schematic diagram below. Figure 3 The first and second recombinant vectors were transfected into HEK293T cells overexpressing TRE3G-GFP at a mass ratio of 1:1. The sgRNA sequence was designed to target the three PAMs TTA, TTC, and TTT in the TRE3G promoter. Activation efficiency was then assessed using FACS. Activation efficiency refers to the percentage of GFP-positive cells.

[0088] 2. The location and type of nuclear localization signals (NLSs) were determined using FACS, and the positional relationship between PmCas12m and VPR was analyzed. A series of PmCas12m-VPR vectors were constructed and transfected into TRE3G-GFPHEK293T cells with sgRNA expression plasmids. Subsequently, the activation efficiency of GFP in the cells was detected by FACS.

[0089] 3. sgRNA plasmids with spacer fragments ranging from 12nt to 26nt were constructed and transfected into HEK293T cells overexpressing TRE3G-GFP along with the #4 version of PmCas12m-VPR plasmid. Activation efficiency was then detected by FACS.

[0090] sgRNA plasmids with spacer fragments of different lengths were constructed using the Golden Gate method.

[0091] When PAM is TTA, spacer fragments of different lengths are as follows:

[0092] 12nt:CTCCCTATCAGT(SEQ ID NO:44);

[0093] 13nt: CTCCCTATCAGTG (SEQ ID NO: 45);

[0094] 14nt: CTCCCTATCAGTGA (SEQ ID NO: 46);

[0095] 15nt: CTCCCTATCAGTGAT (SEQ ID NO: 47);

[0096] 16nt:CTCCCTATCAGTGATA(SEQ ID NO:48);

[0097] 17nt: CTCCCTATCAGTGATAG (SEQ ID NO: 49);

[0098] 18nt: CTCCCTATCAGTGATAGA (SEQ ID NO: 50);

[0099] 19nt: CTCCCTATCAGTGATAGAG (SEQ ID NO: 51);

[0100] 20nt: CTCCCTATCAGTGATAGAGA (SEQ ID NO: 52);

[0101] 21nt: CTCCCTATCAGTGATAGAGAA (SEQ ID NO: 53);

[0102] 22nt: CTCCCTATCAGTGATAGAGAAC (SEQ ID NO: 54);

[0103] 23nt: CTCCCTATCAGTGATAGAGAACG (SEQ ID NO: 55);

[0104] 24nt: CTCCCTATCAGTGATAGAGAACGT (SEQ ID NO: 56);

[0105] 25nt: CTCCCTATCAGTGATAGAGAACGTA (SEQ ID NO: 57);

[0106] 26nt: CTCCCTATCAGTGATAGAGAACGTAT (SEQ ID NO: 58);

[0107] When PAM is TTC, the spacer fragments of different lengths are as follows:

[0108] 12nt:TCTATCACTGAT(SEQ ID NO:59);

[0109] 13nt:TCTATCACTGATA(SEQ ID NO:60);

[0110] 14nt:TCTATCACTGATAG(SEQ ID NO:61);

[0111] 15nt:TCTATCACTGATAGG(SEQ ID NO:62);

[0112] 16nt: TCTATCACTGATAGGG (SEQ ID NO: 63);

[0113] 17nt:TCTATCACTGATAGGGA(SEQ ID NO:64);

[0114] 18nt:TCTATCACTGATAGGGAG(SEQ ID NO:65);

[0115] 19nt:TCTATCACTGATAGGGAGT(SEQ ID NO:66);

[0116] 20nt:TCTATCACTGATAGGGAGTA(SEQ ID NO:67);

[0117] 21nt:TCTATCACTGATAGGGAGTAA(SEQ ID NO:68);

[0118] 22nt:TCTATCACTGATAGGGAGTAAA (SEQ ID NO: 69);

[0119] 23nt:TCTATCACTGATAGGGAGTAAAC (SEQ ID NO:70);

[0120] 24nt:TCTATCACTGATAGGGAGTAAACT(SEQ ID NO:71);

[0121] 25nt:TCTATCACTGATAGGGAGTAAACTG (SEQ ID NO:72);

[0122] 26nt:TCTATCACTGATAGGGAGTAAACTGG (SEQ ID NO:73).

[0123] 4. Each nucleotide of the spacer was subjected to single-point mutation and two consecutive nucleotide mutations, and then transfected into TRE3G-GFPHEK293T cells with the #4 version of the PmCas12m-VPR plasmid. The activation efficiency was then detected by FACS.

[0124] See results Figure 4 The results showed that PmCas12m-VPR significantly activated GFP expression in eukaryotic cells, and the activation effect of GFP expression was superior to that of the TTT PAM sequence when targeting the TRE3G promoter of TTA and TTC PAM sequences. Figure 4 (a) Through optimization of the position and type of nuclear localization signals (NLSs) and analysis of the positional relationship between PmCas12m and VPR, version #4 was found to be the most effective, therefore this vector was selected for subsequent experiments. Figure 4 (b)

[0125] Because the spacer length of sgRNA has a significant impact on the stability and targeting specificity of the sgRNA-DNA complex, this study investigated the effect of spacer sequence length on the epigenetic editing efficiency of the CRISPR-PmCas12m system. The results showed that when the spacer length was 17 nt, PmCas12m-VPR exhibited the best activation effect on GFP. Figure 4(c). Furthermore, to investigate the tolerance to mismatches when sgRNA pairs with the target DNA sequence, each nucleotide of the spacer was subjected to single-point mutations and consecutive two-nucleotide mutations, respectively. Experimental results confirmed that when nucleotides within approximately 14 nt of the 3' end of the crRNA were mutated, the epigenetic editing ability of PmCas12m was almost completely lost. This indicates that the CRISPR-PmCas12m system exhibits high affinity and specificity in epigenetic editing. Figure 4 (d). The above results demonstrate that the CRISPR-PmCas12m system exhibits its application value in the precise regulation of gene expression and epigenetic modification.

[0126] Example 4

[0127] Three-dimensional structural analysis of the PmCas12m-crRNA-target DNA complex

[0128] 1. Recombinant expression and purification of PmCas12m protein

[0129] The PmCas12m expression plasmid with the His6 tag was transformed into E. coli BL21(DE3) strain (TransGen) and cultured overnight at 37°C on LB agar plates containing 50 μg / mL kanamycin. A single colony was picked and inoculated into 10 mL of LB medium containing kanamycin and cultured overnight at 37°C. Subsequently, 10 mL of seed culture was inoculated into 1 LLB-Kan... + In the culture medium, cultured at 37°C until OD 600The pH reached 0.6, and then 0.1 mM IPTG was added to induce protein expression, which was induced overnight at 16°C. Cells were collected by centrifugation, resuspended in buffer A (20 mM HEPES-NaOH, pH 7.6, 20 mM imidazole, 1 M NaCl), and sonicated. The lysis buffer was centrifuged at 15,000 × g for 30 min (4°C), and the supernatant was mixed with 3 mL of Ni affinity resin (Genscript) and incubated at 4°C for 1 h. The mixture was then loaded into an affinity chromatography column (Biosharp), and proteins were eluted using buffer B (20 mM HEPES-NaOH, pH 7.6, 0.3 M imidazole, 0.3 M NaCl). The eluent was then loaded into a pre-equilibrated 5 mL HiTrap Heparin HP column (Cytiva) and eluted with buffer C (20 mM HEPES-NaOH, pH 7.6, 0.3 M NaCl) through a linear gradient of 0.3 to 2 M NaCl. Finally, the protein was purified by gel filtration using a Superdex 200 Increase 10 / 300 column (GE Healthcare) with buffer D (20 mM HEPES-NaOH, pH 7.6, 0.5 M NaCl). The purified protein was stored at -80°C for later use.

[0130] 2.crRNA was transcribed in vitro using the HiScribe T7 HighYieldRNA Synthesis Kit (NEB) and purified using the TransZol Up RNA Extraction Kit (TransGen).

[0131] 3. Cryo-electron microscopy sample preparation and data acquisition

[0132] The PmCas12m2-crRNA-target DNA complex was obtained by recombination of purified PmCas12m, 56nt crRNA, and 36nt target DNA at a molar ratio of 1:1.5:1.5. The ternary complex was purified by gel filtration using a Superdex 200 Increase 10 / 300 column, pre-equilibrated with buffer E (20mM HEPES-NaOH, pH 7.6, 50mM NaCl, 2mM MgCl2, and 10μM ZnCl2).

[0133] Cryo-electron microscopy samples were prepared using a Vitrobot Mark IV (FEI) at 8°C and 100% humidity. Approximately 3 μL of a 3.6 mg / mL complex was added to a glow discharge-treated carbon mesh (Quantifoil 300-mesh gold mesh R1.2 / 1.3). 0.1% OG detergent was added before sample preparation. The mesh was gently aspirated for 2 seconds to remove excess solution before being rapidly immersed in liquid ethane for freezing. Cryo-electron microscopy samples were imaged using a Titan Krios microscope (FEI) at 200 kV using a Falcon 4i detector. Images were automatically acquired using EPU software (Thermo Fisher Scientific) in super-resolution mode, with a nominal magnification of 19,000×, corresponding to the object scale. The pixel size is [not specified], the underfocus range is 0.5 to 2.5 μm, and the total irradiation dose is approximately [not specified].

[0134] 4. Single-particle cryo-electron microscopy data processing

[0135] Cryo-electron microscopy data of the PmCas12m–crRNA–target DNA complex were processed using cryoSPARC software. Initial particle selection was based on 3,793 motion-corrected and dose-weighted micrographs, from which 1,789,514 particles were selected using a blob picker. After multiple rounds of 2D classification, the retained particles were used for heterogeneous refinement, using three ab initio reconstructed images as initial templates. Finally, the selected 121,098 particles underwent non-uniform refinement and local refinement to achieve a final resolution of [resolution missing]. Three-dimensional reconstruction image from cryo-electron microscopy.

[0136] To elucidate the molecular mechanism of PmCas12m, the three-dimensional structure of the PmCas12m-crRNA-target DNA complex was resolved using cryo-electron microscopy. Structural analysis revealed that PmCas12m has a bilobal structure, consisting of a REC lobe and a NUC lobe connected by a linker. The REC lobe contains WED, REC1, and REC2 domains, while the NUC lobe consists of a RuvC domain and a TNB domain. The heteroduplex crRNA-target DNA is located within the positively charged central channel formed by the REC and NUC lobes. PmCas12m recognizes its target through interactions with its sugar-phosphate backbone. Figure 5 ).

[0137] The PmCas12m crRNA contains a 20nt spacer (C1 to A20) and a 33nt crRNA (A(-33) to C(-1)). Figure 5 In the complex structure, the C1 to G17 nucleotides of the crRNA pair with the dC1 to dG17 nucleotides of the target DNA strand (TS), forming a 17 bp sgRNA-target DNA heteroduplex. This indicates that the 17 bp DNA-RNA heteroduplex is the optimal length for PmCas12m-mediated DNA binding, which also explains the experimental results showing optimal activity when the sgRNA spacer length is 17 nt. In the crRNA scaffold region, G(-28) to C(-26) and G(-15) to C(-17) form classic Watson-Crick base pairings, while C(-23) to C(-20) and G(-3) to G(-6) pair, collectively stabilizing the crRNA conformation. The WED and RuvC domains jointly recognize the crRNA, nucleotide A(-29) is linked to Lys361 and Arg457 via hydrogen bonds, and the phosphate backbone of G(-28) forms a hydrogen bond with Lys468 of the RuvC domain. Furthermore, the nucleotides U(-22), G(-6), and G(-3) through C1 extensively interact with multiple residues (including Arg228, Arg230, Arg262, Asp475, Lys540, and Arg541) in the WED and RuvC domains. Simultaneously, the phosphate backbones of G9, A10, and T11 in the TS chain bind to Ser283, Arg466, and Arg473, respectively. In the PAM region, the non-target strand (NTS) sequence of 5′-TTA-3′PAM interacts with the REC1 domain: the phosphate backbones of dT(-3*) and dT(-2*) form hydrogen bonds with Tyr166 and Tyr156, respectively, while dT(-2*) also binds to Asn167 in the REC1 domain. These structural features reveal the molecular mechanism by which PmCas12m specifically recognizes 5′-TTA-3′PAM.

[0138] Unlike most Cas12 family enzymes, the RuvC active site of PmCas12m possesses a non-classical catalytic motif. The classic DED catalytic residues are replaced by Asn372, Asp494, and Asp581 within the RuvC domain (i.e., the NDD motif). Furthermore, dG(11*) to dG(13*) nucleotides of the NTS chain are located near this non-classical NDD active site. It is speculated that the NDD motif of PmCas12m may lack magnesium ion interaction, leading to the loss of target DNA cleavage activity. In summary, the cryo-electron microscopy structure of the PmCas12m-crRNA-target DNA complex provides crucial structural evidence for elucidating the substrate recognition and interaction patterns of this protein.

[0139] Example 5

[0140] Structure-guided PmCas12m protein mutation optimization

[0141] To enhance the epigenetic editing activity of PmCas12m, saturation mutagenesis (DMS) was employed to investigate how amino acid mutations affect the epigenetic editing efficiency of PmCas12m. By analyzing the structure of the PmCas12m-crRNA-target DNA complex, 137 residues were selected for saturation mutagenesis based on amino acid accessibility and their proximity to crRNA and target DNA. Figure 6 (a, c). These residues were divided into four regions: residues 121-185, residues 226-264, residues 306-315, and residues 493-515. For each region, a PmCas12m-VPR mutant library was constructed, in which each amino acid was mutated to one of the other 19 amino acids, thus covering all 20 possible amino acid substitutions. Figure 6 (c)

[0142] To avoid cross-contamination, the four libraries were packaged into lentiviruses and transduced into HEK293T cells containing TRE3G-GFP that stably expressed the corresponding sgRNAs (DNA sequences corresponding to sgRNAs with PAM of TTA: CTCCCTATCAGTGATAG, SEQ ID NO: 74; DNA sequences corresponding to sgRNAs with PAM of TTC: TCTATCACTGATAGGGA, SEQ ID NO: 75). The MOI (multiple of infection) at infection was controlled below 0.3 to ensure that only one PmCas12m mutant was expressed in each cell. 72 hours post-infection, high-GFP and low-GFP expression cells were sorted by flow cytometry (FACS) and cultured for 5 days respectively. Subsequently, high-GFP and low-GFP expression cells were sorted again by flow cytometry. Approximately 1 million cells were collected from each group. Total RNA was extracted from each group of cells, and cDNA was synthesized. Each library was then amplified using primers containing Illumina sequencing adapters and barcodes for Illumina HiSeq sequencing (PE150). The abundance of each mutant was counted and normalized to the total sequencing reads for that group. Activity was calculated as the ratio of the corresponding mutant sequencing reads in the high-GFP expression group to those in the low-GFP expression group.

[0143] Based on structural analysis and DMS screening results, four mutations (S128Q, A143R, A146C, and E147H) were found to be significantly superior to the control group in epigenetic editing efficiency, as they were located near the nucleic acid binding site. Figure 6 (d, e).

[0144] To further enhance the activity of PmCas12m, a second round of optimization was performed in this embodiment. In this round of optimization, the four mutant residues were combined in pairs to construct six double mutants. The activity of all double mutants was superior to that of the single mutants optimized in the first round. In the third round of screening, we constructed three triple mutants and one quadruple mutant based on the results of the second round of screening. Ultimately, the A143R / A146C / E147H triple mutant exhibited the highest epigenetic editing activity, so we named it PmCas12m-RCH( Figure 6 (d, e).

[0145] Epigenetic editing tools typically require the fusion of multiple effector domains. Even with compact proteins like PmCas12m-RCH, their overall molecular weight remains relatively large, posing a challenge to in vivo delivery efficiency. Therefore, reasonable protein size reduction could significantly improve delivery efficiency, thereby promoting the development of efficient epigenetic editing. To this end, based on structural analysis of PmCas12m, five potential deletion regions were screened. These regions are either relatively flexible or unrelated to DNA / RNA binding. Figure 6 (f). We found that the PmCas12m-RCH variant with simultaneous deletions of the N-terminus (Δ2-13) and C-terminus (Δ594-601) still maintained epigenetic editing activity close to that of PmCas12m-RCH, and we named it PmCas12m-RCH-V1. Figure 6 (g). However, when we further deleted the WED (Δ285-299), RuvC (Δ382-450), or TNB (Δ551-578) regions individually from PmCas12m-RCH-V1, the editing activity of the resulting mutants PmCas12m-RCH-V2, PmCas12m-RCH-V3, and PmCas12m-RCH-V4 was significantly reduced or even completely lost. Figure 6 (g).

[0146] Example 6

[0147] Epigenetic editing capability test of xCas12m

[0148]

[0149] sgHBG: TTCCGATTCAGTCATTC (SEQ ID NO: 19);

[0150] sgIL1RN: AGTCACCCTCCTGGAAA (SEQ ID NO: 20);

[0151] sgTTN: AGTAAGTAGCCAGGTCT (SEQ ID NO: 21);

[0152] sgASCL1:CAAGGAGCGGGAGAAAG (SEQ ID NO: 22);

[0153] sgFOXA3: TATAGCCGGGACACCCC (SEQ ID NO: 23);

[0154] sgCD34:AACGAGGCATCTGGAGC (SEQ ID NO: 24);

[0155] sgIL2RA:TTATGGGCGTAGCTGAA (SEQ ID NO: 25).

[0156] xCas12m-VPR and its corresponding sgRNA plasmid were transfected into HEK293T cells. After 72 hours, total RNA was extracted from the cells using the TransZol Up RNA Extraction Kit (TransGen). cDNA synthesis was performed using TransScript cDNA Synthesis SuperMix (TransGen). qRT-PCR reactions were prepared in 96-well plates using PerfectStartGreen qPCR SuperMix (TransGen). To assess the epigenomic activation efficiency of xCas12m-KRAB-MeCP2, the expression levels of seven endogenous genes in HEK293T cells were measured using qRT-PCR. xCas12m-KRAB-MeCP2 and its corresponding sgRNA plasmid were transfected into HEK293T cells. After 72 hours, total RNA was extracted from the cells using the TransZol Up RNA Extraction Kit (TransGen). cDNA synthesis was performed using TransScript cDNA Synthesis SuperMix (TransGen). qRT-PCR reactions were prepared in 96-well plates using PerfectStart Green qPCR SuperMix (TransGen).

[0157] The primers used for qRT-PCR detection are as follows:

[0158] HBG-F:TGGATGATTCTCAAGGGCAC(SEQ ID NO:76);

[0159] HBF-R: TCAGTGGTATCTGGAGGACA (SEQ ID NO: 77);

[0160] IL1RN-F:CATTGAGCCCTCATGCTCTGTT(SEQ ID NO:78);

[0161] IL1RN-R:CACTGTCTGAGCGGATGAA(SEQ ID NO:79);

[0162] TTN-F:TGTTGCCACTGGTGCTAAAG(SEQ ID NO:80);

[0163] TTN-R:ACAGCAGTCTTCTCCGCTTC(SEQ ID NO:81);

[0164] ASCL1-F:GGAGCTTCTCGACTTCACCA(SEQ ID NO:82);

[0165] ASCL1-R:AACGCCACTGACAAGAAAGC(SEQ ID NO:83);

[0166] FOXA3-F:GAGATGCCGAAGGGGTATCG(SEQ ID NO:84);

[0167] FOXA3-R: TGATTCTCCCGGTAGTAAGGG(SEQ ID NO:85);

[0168] CD34-F:AATAGCCAGTGATGCCCAAG(SEQ ID NO:86);

[0169] CD34-R:GGTATGCTCCCTGCCTCCTT(SEQ ID NO:87);

[0170] IL2RA-F:AGGGATACAGGGCTCTACAC(SEQ ID NO:88);

[0171] IL2R2-R:TGGTCTCCATTTCACCTGTG(SEQ ID NO:89);

[0172] CXCR4-F:ACTACACCGAGGAAATGGGCT(SEQ ID NO:90);

[0173] CXCR4-R:CCCACAATGCCAGTTAAGAAGA(SEQ ID NO:91);

[0174] B2M-F:CATGTAAGCAGCATCATGGAG(SEQ ID NO:92);

[0175] B2M-R:CCCTACATTTTGTGCATAAAGTG(SEQ ID NO:93);

[0176] RBM3-F:TGACCGCTACTCAGGAGGAA(SEQ ID NO:94);

[0177] RBM3-R:CTTCGGTGCAGCTCCAAAA(SEQ ID NO:95);

[0178] HINT1-F:TTTCCCCTCAAGCACCAACA(SEQ ID NO:96);

[0179] HINT1-R:ATTCAGGCCCAGATCAGCAG(SEQ ID NO:97);

[0180] CHEK1-F:ATATGAAGCGGTGCCGTAGACT(SEQ ID NO:98);

[0181] CHEK1-R:TGCCTATGTCTGGCTCTATTCTG(SEQ ID NO:99);

[0182] BRCA1-F:CTCAAGGAACCAGGGATGAA(SEQ ID NO:100);

[0183] BRCA1-R:GCTGTAATGAGCTGGCATGA(SEQ ID NO:101);

[0184] MET-F:ATCAGAGGGTCGCTTCATGC(SEQ ID NO:102);

[0185] MET-R:GGATCTTCGTGATCTTCTTCCCA(SEQ ID NO:103);

[0186] GAPDH-F: CTGGGCTACACTGAGCACC (SEQ ID NO: 104);

[0187] GAPDH-R: AAGTGGTCGTTGAGGGCAATG (SEQ ID NO: 105).

[0188] Qrt-PCR was performed using PerfectStart Green qPCR SuperMix (TransGen). The reaction mixture was prepared as follows: 10 μl PerfectStart Green qPCR SuperMix (TransGen), 1 μl DNA template, 1 μl upstream primer (5 μM), 1 μl downstream primer (5 μM), and ddH2O to a final volume of 20 μl. The reaction program was: pre-denaturation at 95℃ for 3 min; cycling at 95℃ for 5 sec, 60℃ for 30 sec, and 72℃ for 30 sec for 40 cycles. Amplification results were analyzed using 2... -ΔΔCt Legal analysis.

[0189] See results Figure 7 The results showed that both wild-type PmCas12m-VPR and xCas12m-VPR could efficiently and stably activate these endogenous genes, with xCas12m-VPR consistently showing higher activation efficiency than wild-type PmCas12m-VPR. This further demonstrates the effectiveness and practicality of our protein optimization strategy in enhancing epigenetic editing activity. Figure 7 (bh). Notably, on some genes, the activation capacity of xCas12m-VPR is even superior to that of the widely used dCas9-VPR, demonstrating its great potential in epigenetic editing applications. Figure 7 (middle bd).

[0190] Given the immense potential of xCas12m in CRISPR-mediated gene activation, we further explored its broader applications in epigenetic editing. To this end, we employed the classic transcriptional repression effector protein KRAB-MeCP2 (a widely used effector protein for CRISPR-mediated gene silencing) and fused it with the aforementioned eight CRISPR-Cas systems. Figure 7 (i). Same as the activation experiment. xCas12m-KRAB-MeCP2 showed varying degrees of gene repression in all seven endogenous genes. Figure 7 The gene silencing efficiency of xCas12m-KRAB-MeCP2 is comparable to that of dCas9-KRAB-MeCP2 and its variant dSpRY-KRAB-MeCP2. Figure 7In some cases, the inhibition efficiency of xCas12m-KRAB-MeCP2 is even higher than that of dCas9-KRAB-MeCP2. Figure 7 The results suggest that xCas12m-KRAB-MeCP2 may be an effective alternative to the CRISPRi (CRISPR-mediated gene repression) strategy.

[0191] In summary, xCas12m has the potential for efficient and flexible epigenome editing, laying the foundation for epigenetic regulation research and therapeutic gene regulation applications.

[0192] Example 7

[0193] xCas12m Inhibition of HBV Infection in Cells Experiment

[0194] To evaluate the interference efficiency of xCas12m-CRISPRoff in vitro, three different HBV cell models were used: (1) HepG2 cells transfected with 1.3×HBV plasmid; (2) HepAD38 cell line, which integrates the HBV genome and expresses HBV under tetracycline control; and (3) HBV-infected HepG2-NTCP cells. Nine sgRNAs (see Table 1) were designed to target four viral promoters and two viral enhancers in the HBV genome to interfere with key regulatory elements essential for HBV transcription and replication.

[0195] Table 1. DNA sequences corresponding to sgRNAs targeting the HBV genome.

[0196] sg2 CCCCGCTGTCTCCACCT(SEQ ID NO:27) sg3 CGCAGTATGGATCGGCA(SEQ ID NO:28) sg4 CATAAGAGGACTCTTGG(SEQ ID NO:29) sg5 CGCACCAGCACCATGCA(SEQ ID NO:30) sg6 CAAGGCCTTTCTGTGTA(SEQ ID NO:31) sg7 AAGACTGGGAGGAGTTG(SEQ ID NO:32) sg8 TATAATATACCCGCCTT(SEQ ID NO:33) sg9 TGTAAGACCTTGGGCAA(SEQ ID NO:34)

[0197]

[0198] Construction method of sgRNA plasmid: The sgRNA fragment was constructed into the BsmBI site of the pSLQ9834(Addgene#176272) plasmid using the Golden Gate method.

[0199] The xCas12m-CRISPRoff plasmid and sgRNA plasmids targeting HBV genome promoter or enhancer sequences were co-transfected into three different HBV cell models. Cells were collected after 3 and 5 days of culture. Cell supernatants were collected. HBeAg and HBsAg levels in the cell supernatant were measured using an ELISA kit (Autobio). The results showed that all sgRNAs led to a decrease in HBeAg and HBsAg levels, although the interference efficiency of different sgRNAs varied. Figure 8 (c)

[0200] Subsequently, the antiviral effect was further evaluated using high-efficiency sgRNA. Specifically, the xCas12m-CRISPRoff plasmid and sgRNA plasmids (sg1, sg2, sg6, sg7, sg9) targeting HBV genome promoter or enhancer sequences were co-transfected into three different HBV cell models. Cells were collected at different time points, and total RNA was extracted from cells at different time points using the TransZol Up RNA Extraction Kit (TransGen). cDNA synthesis was performed using TransScript cDNA Synthesis SuperMix (TransGen). qRT-PCR was used to detect HBV total RNA and pgRNA levels to analyze viral transcription and replication. HBV total RNA reflects the overall viral transcription level, while pgRNA is a key intermediate in viral genome replication. qRT-PCR reactions were prepared in 96-well plates using PerfectStart Green qPCR SuperMix (TransGen). The qRT-PCR primers were:

[0201] HBV total RNA-F: GAGTGTGGATTCGCACTCC (SEQ ID NO: 106);

[0202] HBV total RNA-R: GAGGCGAGGGAGTTCTTCT (SEQ ID NO: 107);

[0203] pgRNA-F: TCACCAGCACCATGCAAC (SEQ ID NO: 108);

[0204] pgRNA-R: AAGCCACCCAAGGCACAG (SEQ ID NO: 109).

[0205] The results showed that the levels of total HBV RNA and pgRNA were significantly reduced. Figure 8 The results (df) indicate that xCas12m-CRISPRoff can effectively interfere with HBV transcription and replication.

[0206] The xCas12m-CRISPRoff plasmid, along with the sg1 and sg9 plasmids, was transfected into HepG2-NTCP cells, and RNA-seq sequencing was performed at specified time points. RNA-seq analysis further confirmed that xCas12m-CRISPRoff significantly reduced the abundance of HBV transcripts in HBV-infected HepG2-NTCP cells. Figure 8 (g).

[0207] Finally, this embodiment detected the levels of total HBV DNA and cccDNA in HBV-infected HepG2-NTCP cells. Total HBV DNA represents the overall viral genome, while cccDNA, as the circular genome of HBV, is crucial for viral DNA replication. The xCas12m-CRISPRoff plasmid and sgRNA plasmids targeting HBV genome promoter or enhancer sequences were co-transfected into three different HBV cell models, and the supernatant was extracted for quantitative detection of total HBV DNA. In short, HBV DNA was extracted from cell pellets, supernatants, or serum, and quantified using the TIANamp Viral DNA / RNA Extraction Kit (TIANGEN) via qRT-PCR. The equivalent copy number of the viral genome was calculated using a 2×HBV plasmid standard curve with a concentration range of 10-1. 1 Up to 10 9For the detection of cccDNA (covalently closed circular DNA), extracted DNA samples were treated with Plasmid-safe DNase (Lucigen) at 37°C for 30 minutes to degrade linear DNA, followed by heat inactivation at 70°C for 30 minutes. The reaction products were then quantified by qRT-PCR, and the equivalent viral genome copy number was calculated from a standard curve generated based on known copy numbers. HBV total DNA-F: CCGTCTGTGCCTTCTCATCTG (SEQ ID NO: 110); R: AGTCCAAGAGTCCTCTTATGTAAGACCTT (SEQ ID NO: 111). cccDNA-F: TGCACTTCGCTTCACCT (SEQ ID NO: 112); cccDNA-R: AGGGGCATTTGGTGGTC (SEQ ID NO: 113).

[0208] The results showed that xCas12m-CRISPRoff significantly reduced the levels of total HBV DNA and cccDNA, indicating that xCas12m-CRISPRoff can effectively interfere with the maintenance of the HBV genome. Figure 8 (Chinese hi).

[0209] Example 8

[0210] xCas12m inhibits HBV infection in animals.

[0211] Preparation methods of AAV-xCas12m-CRISPRoff-sg1 or AAV-xCas12m-CRISPRoff-sg9:

[0212] 1. Co-transfect the prepared plasmid DNA with the helper plasmid required for AAV packaging into the cells.

[0213] 2. After 5-7 days of culture, crude AAV virus is collected. The cell supernatant and cell pellet are collected by centrifugation, and repeated freeze-thaw cycles are performed to separate the virus particles from cell debris and other impurities. Next, AAV is purified by centrifugation and ultrafiltration to remove residual cellular proteins and DNA.

[0214] 3. The purified AAV samples were subjected to concentration determination and quality testing to ensure that the viral yield and quality met the requirements.

[0215] Six-week-old C57BL / 6 mice were injected via the tail vein with 1×10 10The AAV-HBV1.04 of vg (Xu Z, Zhao L, Zhong Y, et al. A Novel Mouse Model Harboring Hepatitis B Virus Covalently Closed Circular DNA. CellMol Gastroenterol Hepatol. 2022; 13(4):1001-1017. doi:10.1016 / j.jcmgh.2021.11.011). Seven days later, the mice were randomly divided into three groups and injected with 1×10 11 The HBV DNA, HBeAg, and HBsAg levels were determined using AAV-xCas12m-CRISPRoff-sgNT (sgNT: CGAATACGCCCACGCGA, SEQ ID NO: 122), AAV-xCas12m-CRISPRoff-sg1, or AAV-xCas12m-CRISPRoff-sg9. Blood samples were collected at predetermined time points, and serum was obtained by centrifugation at 1500×g for 15 minutes (4°C). This serum was used to detect the levels of HBV DNA, HBeAg, and HBsAg. The detection method was the same as described in Example 7.

[0216] Due to the compact structure of xCas12m-CRISPRoff (1305 amino acids), the xCas12m-CRISPRoff system and its sgRNA component were successfully engineered into a single AAV vector. Figure 7 (j). This configuration is well-suited to the packaging limitations of AAV vectors and may facilitate efficient in vivo delivery, opening up possibilities for potential treatments of HBV infection.

[0217] The test results showed that xCas12m-CRISPRoff significantly reduced serum HBeAg levels, HBsAg levels, and circulating HBV DNA levels. Figure 8 (ln). Notably, the reduction in viral products was observed to be persistent after a single dose. Figure 8 The results (ln) are attributed to the genetic silencing memory of CRISPRoff. Overall, these results highlight xCas12m-CRISPRoff as a promising epigenome editing tool for suppressing HBV antigen expression and circulating HBV DNA, thus providing new insights for developing epigenetic silencing therapies against HBV infection.

[0218] In summary, xCas12m-CRISPRoff exhibited significant antiviral activity in multiple HBV cell models. By targeting key transcriptional regulatory elements in HBV, it effectively inhibited viral transcription and replication, and reduced viral DNA load. These results demonstrate that xCas12m-CRISPRoff is a highly efficient and safe epigenetic editing tool, providing a new strategy for HBV treatment.

[0219] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A PmCas12m protein mutant for epigenetic editing, characterized in that, The PmCas12m protein mutant is xCas12m; the amino acid sequence of xCas12m is shown in SEQ ID NO:

2.

2. A fusion protein, characterized in that, It is formed by fusing the PmCas12m mutant of claim 1 with an epigenetic effect domain, wherein the epigenetic effect domain includes a transcriptional repression domain and / or a transcriptional activation domain. The transcriptional activation domain is the VP64-P65AD-Rta effector; The transcriptional repression domain is KRAB-MeCP2 and / or CRISPRoff.

3. A CRISPR-Cas12m system for epigenetic editing, characterized in that, It includes at least one protein selected from the PmCas12m protein mutant of claim 1 or the fusion protein of claim 2.

4. A vector composition for epigenetic editing, characterized in that, This includes a first recombinant vector containing sgRNA and a second recombinant vector containing a nucleic acid molecule for encoding a protein; The protein includes at least one of the PmCas12m protein mutant of claim 1 or the fusion protein of claim 2.

5. The carrier composition according to claim 4, characterized in that, The first recombinant vector containing sgRNA also includes a spacer fragment; The length of the spacer segment is 16~20nt.

6. The use of the PmCas12m protein mutant of claim 1, the fusion protein of claim 2, the CRISPR-Cas12m system of claim 3, or the vector composition of claim 4 or 5 in the preparation of an antiviral drug.

7. The application according to claim 6, characterized in that, The virus mentioned includes the hepatitis B virus.

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