Application of methyltransferase mutant and deaminase in DNA methylation single base resolution sequencing

Abstract

The invention discloses application of a methyltransferase mutant and deaminase in DNA methylation single base resolution sequencing, and relates to the technical field of gene sequencing. On the basis of a methyltransferase mutant M.MedI-N377K, cytosine at a CG site in DNA (Deoxyribonucleic Acid) can be specifically recognized, and carboxymethyl of carboxyl-S-adenosine-L-methionine (caSAM) is transferred to a C5 site of C in the CG, so that the 5-carboxymethyl cytosine is generated. 5-methylcytosine can be subjected to deamination by deaminase to form thymine, and the thymine is paired with adenine in subsequent PCR amplification; and 5cam C can resist deamination of A3A and is paired with guanine in subsequent PCR (Polymerase Chain Reaction), and 5mC is read as T and C is read as C in a subsequent Sanger sequencing result of an amplification product, so that C and 5mC in DNA (Deoxyribonucleic Acid) are distinguished. The method disclosed by the invention is high in sensitivity, good in specificity and simple to operate, does not involve hydrosulfite treatment, and can be used for carrying out positioning analysis on 5mC in a biological sample under a single-base resolution ratio.

Description

Technical Field

[0001] The present invention relates to the technical field of gene sequencing, and particularly relates to an application of a methyltransferase mutant and a deaminase in single-base resolution sequencing of DNA methylation. Background Art

[0002] DNA methylation is a key epigenetic process and is conserved in most higher eukaryotes. In mammals, DNA methylation mainly occurs at cytosine in CpG dinucleotides, mainly by adding a methyl group at the C5 position of cytosine (C) in CG dinucleotides by DNA methyltransferase to produce 5-methylcytosine (5mC), which is the most widespread epigenetic modification in mammals and is also called the "fifth base" of DNA. 5mC plays an important role in the regulation of gene expression and the occurrence of many diseases. The accurate positioning of 5mC has far-reaching significance for studying its biological functions.

[0003] Currently, bisulfite sequencing (BS-seq) is still regarded as the gold standard for 5mC detection. Its basic principle is: treating the DNA to be sequenced with bisulfite under acidic conditions, unmodified C will be deaminated to form uracil (U), while 5mC will not be affected by bisulfite, and C and 5mC are read as T and C respectively during the sequencing process. However, the chemical reaction conditions of bisulfite are severe, which can degrade up to 99% of DNA, severely limiting the application of this method. Secondly, BS-seq depends on completely converting unmodified C into U, which will greatly reduce the sequence complexity, resulting in poor sequencing quality and increased cost.

[0004] In addition, there are also epigenetic sequencing technologies relying on biological enzymes, such as the TET dioxygenase-assisted pyridine borane sequencing method (TAPS). In TAPS, 5mC is oxidized to 5-carboxylcytosine (5caC) by TET protein, and then treated with pyridine borane. 5caC can be reduced to dihydrouracil (DHU), which pairs with A during the subsequent PCR process and is read as T during the sequencing process, so it can be used for single-base resolution detection of 5mC. However, it is difficult for TET dioxygenase in this method to completely oxidize 5mC to 5caC.

[0005] With the discovery of enzymology research, the AID / APOBEC family deaminase APOBEC3A (human apolipoprotein B mRNA editing enzyme catalytic subunit 3A, A3A) can recognize substrates C and 5mC, and has relatively low deamination activity towards 5caC. Based on this, a combined enzymatic methylation sequencing method (EM-seq) of A3A, TET protein, and β-glucosyltransferase was developed. In the EM-seq sequencing method, first, the TET protein and β-glucosyltransferase are used to transform and protect 5mC and 5-hydroxymethylcytosine, and then the A3A deaminase is added for treatment. During this process, the protected 5mC and 5hmC antagonize the deamination of A3A and are read as C during sequencing, while C will be deaminated and read as T. Similar to the TET protein oxidation function mentioned in the TAPS method, the TET protein cannot completely convert 5mC to 5caC, resulting in false-positive results. In addition, EM-seq involves three different biological enzymes, and the operation is complex and time-consuming.

[0006] The mutant of the CG site-specific methyltransferase M.MpeI (M.MpeI-N374K) can transfer the carboxymethyl group on caSAM to the C5 position of C in the CG site to form 5-carboxymethylcytosine (5camC). 5camC can antagonize the deamination of A3A and is read as C in Sanger sequencing, while 5mC will be deaminated by the A3A protein and read as T during sequencing. Based on this, a sequencing method for localizing 5mC in DNA by methyltransferase labeling and A3A deamination sequencing (MLAD-seq) was derived. However, in the MLAD-seq sequencing method, the labeling efficiency of M.MpeI-N374K is easily restricted by the opposite strand. The direct sequencing method of DNA methylation attempts to solve this problem. This method can achieve the localization analysis of methylation, but it requires one-step extension of DNA and then carboxymethylation of all Cs, and the operation is complex.

[0007] In summary, if a methyltransferase with higher carboxymethyl labeling efficiency can be discovered or obtained, 5mC can be analyzed for localization with simpler operations, while reducing the false-positive rate of 5mC analysis. Summary of the Invention

[0008] The present invention provides an application of a methyltransferase mutant and a deaminase in single-base resolution sequencing of DNA methylation. The present invention combines the methyltransferase mutant M.MedI-N377K and the deaminase A3A to realize a resolution localization sequencing analysis method for methylated cytosine (5mC) at CG sites in the DNA to be sequenced. It can be widely applied in the field of DNA modification sequencing and has the characteristics of high sensitivity, high selectivity, and simple operation. Specifically, it is achieved through the following technologies.

[0009] In the first aspect of the present invention, there is provided an application of a methyltransferase mutant and a deaminase in single-base resolution sequencing of DNA methylation. Using the methyltransferase mutant M.MedI-N377K and carboxy-S-adenosyl-L-methionine, the original DNA to be sequenced is subjected to carboxymethylation treatment;

[0010] Denaturation treatment is carried out to obtain single-stranded carboxymethylated DNA to be sequenced;

[0011] The single-stranded carboxymethylated DNA to be sequenced is subjected to deamination treatment using the deaminase. After inactivating the methyltransferase mutant and the deaminase, amplification and sequencing are carried out, and the sequencing results are compared with the sequencing results of the original DNA to be sequenced to obtain the sites of 5-methylcytosine in the CG sites of the original DNA to be sequenced;

[0012] The amino acid sequence of the methyltransferase mutant M.MedI-N377K is shown in SEQ ID NO.1.

[0013] The methyltransferase mutant M.MedI-N377K provided by the present invention has a higher carboxymethylation labeling efficiency for cytosine (C) in the CG sites of the DNA to be tested, and can specifically transfer carboxymethyl to the C5 position of C in the CG sites to generate 5-carboxymethylcytosine (5camC), while methylated cytosine is not affected. 5camC can resist the deamination of the deaminase (A3A), while methylated cytosine (5mC) will be deaminated by A3A. In the final Sanger sequencing, the deaminated 5mC is read as T, and 5camC is read as C.

[0014] Based on the above characteristics of the methyltransferase mutant M.MedI-N377K and methylated cytosine (5mC). In the present invention, the C in the CG sites of the DNA to be sequenced is subjected to carboxymethylation treatment by the methyltransferase mutant M.MedI-N377K, and then combined with A3A deaminase for deamination treatment. 5camC antagonizes the deamination of A3A, while 5mC is deaminated by A3A; sequencing is carried out to obtain the single-base resolution localization information of 5mC in the original CG sites.

[0015] Specifically, by comparing the sequencing results of the original DNA to be sequenced and the sequencing results after the above treatment, find the sites that are read as CG in the original DNA to be sequenced and become TG sites, then the T site here is the 5mC site in the original DNA to be sequenced.

[0016] Furthermore, the gene sequence encoding the methyltransferase mutant M.MedI-N377K is shown in SEQ ID NO.2.

[0017] Furthermore, the amino acid sequence of the deaminase is shown in SEQ ID NO.5.

[0018] Furthermore, the gene sequence encoding the deaminase is as shown in SEQ ID NO.6.

[0019] In the second aspect of the present invention, a methyltransferase mutant M.MedI-N377K is provided, and its amino acid sequence is as shown in SEQ ID NO.1.

[0020] Furthermore, the gene sequence encoding the methyltransferase mutant M.MedI-N377K is as shown in SEQ ID NO.2.

[0021] In the third aspect of the present invention, a biological material is provided, and the biological material is any one of the following:

[0022] (1) A nucleic acid molecule for encoding the above-mentioned methyltransferase mutant M.MedI-N377K;

[0023] (2) A recombinant vector, which contains a gene fragment for encoding the above-mentioned methyltransferase mutant M.MedI-N377K;

[0024] (3) An engineered cell, which includes the nucleic acid molecule or the recombinant vector.

[0025] In the fourth aspect of the present invention, a single-base resolution sequencing analysis method for 5-methylcytosine at the CG site of a DNA to be tested is further provided, including the following steps:

[0026] Using the above-mentioned methyltransferase mutant M.MedI-N377K and carboxy-S-adenosyl-L-methionine to perform carboxymethylation treatment on the original DNA to be sequenced;

[0027] Performing denaturation treatment to obtain single-stranded carboxymethylated DNA to be sequenced;

[0028] Using a deaminase to perform deamination treatment on the single-stranded carboxymethylated DNA to be sequenced, inactivating the methyltransferase mutant and the deaminase, and then amplifying and sequencing;

[0029] Comparing the sequencing result with the sequencing result of the original DNA to be sequenced to obtain the site of 5-methylcytosine at the CG site of the original DNA to be sequenced.

[0030] Optionally, in the above method, in the buffer used for performing carboxymethylation treatment on the original DNA to be sequenced, the final concentrations of each component are 10-15 mM tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), 50-60 mM sodium chloride, 1-2 mM dithiothreitol, and 1-2 mM ethylenediaminetetraacetic acid, and the pH is 7.5-8.0.

[0031] Based on the action of the methyltransferase mutant M.MedI-N377K, the specific dosage and reaction conditions are such that the C at the CG site in the DNA can be fully carboxymethylated and labeled.

[0032] Optionally, in the above method, the conditions for carboxymethylating the original DNA to be sequenced are reacting at 37 °C for 1 - 2 h.

[0033] Optionally, in the above method, the method for denaturing the carboxymethylated original DNA to be sequenced is: incubating the DNA at a high temperature of 90 - 95 °C for 10 - 15 min and then immediately transferring it to an ice bath for 5 - 10 min to denature the double-stranded DNA into single-stranded DNA.

[0034] Optionally, in the above method, when performing deamination treatment on the single-stranded carboxymethylated DNA to be sequenced, the final concentrations of the components in the deamination reaction buffer used are 20 - 25 mM of 2-morpholinoethanesulfonic acid and 0.1 - 0.2% of TritonX-100, and the pH is 6.5 - 7.0.

[0035] Based on the action of the deaminase, the specific dosage and reaction conditions are such that the C at the CG site in the DNA can be fully carboxymethylated and labeled.

[0036] Optionally, the reaction conditions for deamination treatment are reacting at 37 °C for 3 - 5 h.

[0037] Optionally, the conditions for inactivating the methyltransferase mutant and the deaminase are incubating at 90 - 95 °C for 10 - 15 min. After the reaction temperature cools to room temperature, subsequent operations can be carried out.

[0038] Furthermore, in the method for analyzing the localization of 5-methylcytosine at the CG site of the DNA to be tested above, the concentration of carboxy-S-adenosyl-L-methionine is 200 - 400 μM.

[0039] In the fifth aspect of the present invention, there is provided a product for single-base resolution sequencing analysis of 5-methylcytosine at the CG site of DNA to be tested, and the product includes the above-mentioned methyltransferase mutant M.MedI-N377K and a deaminase.

[0040] Compared with the prior art, the advantages of the present invention are as follows:

[0041] 1. The methyltransferase mutant M.MedI-N377K provided by the present invention, and the method and corresponding product for analyzing the localization of 5-methylcytosine at the CG site of DNA by combining it with a deaminase are simple to operate, without a cumbersome sample pretreatment process, nor a bisulfite treatment and subsequent purification process.

[0042] 2. The methyltransferase mutant M.MedI-N377K provided by the present invention has a very high carboxymethylation efficiency for C in the CG site of the DNA to be tested.

[0043] 3. The deaminase involved in the present invention has a high deamination efficiency for 5-methylcytosine (>98%), while the deamination efficiency for 5-carboxymethylcytosine is relatively low (<1%), which is very beneficial for the single-base resolution mapping analysis of 5mC. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 It is a schematic diagram of the localization process of 5-methylcytosine in DNA by using the methyltransferase mutant M.MedI-N377K in combination with the deaminase A3A in the present invention.

[0045] Figure 2 It is the LC-MS / MS detection effect before and after the carboxymethylation treatment of a 28-bp DNA strand containing CG sites synthesized artificially by M.MedI-N377K.

[0046] Figure 3 It is the sequencing effect diagram of the DNA strands (DNA-C, DNA-5mC) containing C and 5mC synthesized artificially after the carboxymethylation treatment by M.MedI-N377K and the deamination treatment by A3A. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0047] The technical solutions of the present invention will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0048] Example 1: Preparation, synthesis and property analysis of the methyltransferase mutant

[0049] The specific steps are as follows:

[0050] (1) The amino acid sequence of the wild-type methyltransferase M.MedI used in the present invention refers to the National Center for Biotechnology Information (NCBI accession: WP_270161023.1). Site-directed mutagenesis is performed on it, and asparagine (N) at position 377 is mutated to lysine (K) to obtain a protein mutant (M.MedI-N377K) with higher carboxymethylation efficiency.

[0051] The gene sequence encoding the methyltransferase mutant M.MedI-N377K is inserted into the pET-28a(+) plasmid vector, and the C-terminus of the protein of the methyltransferase mutant carries a His tag to construct the corresponding recombinant expression vector.

[0052] The recombinant expression vector was transformed into Escherichia coli BL21(DE3)pLysS cells for protein expression of the methyltransferase mutant M.MedI-N377K.

[0053] After protein expression, purification was performed using His magnetic beads to obtain the full-length methyltransferase mutant M.MedI-N377K protein.

[0054] The amino acid sequence of the methyltransferase mutant M.MedI-N377K is as follows:

[0055] MKEIKIFEFFSGIGSQMKALKNLEKSLNFTTKSVGACDFYIDAIVSYMCIHHGNLEPENDFTKEEMISILDKFKFSNNSKDIVSENYFKKINEQKLRQLFPYLFAFINNDYFNKKYDKNILQYERLNATDIRDFDTLPDNIDILTYSFPCQDLSQQGKQKGIEKNTRSGLLYEIERILKLNLNNLPKVLILENVKALVSKKFINQFNAWINVLSELGYKSSWKIMNASDFGSAQNRERVFMVSVLSDENFEFPKINENNSKNVSNIWEYDGEHKIIQLDSNQKMNDFKITKNKIQKAFINNYSNFNSENYIYSINSKGATLTASGANSRLKFWVNNEIQIMNSLEALLYMGFERDDYEKIKSSNLLNENKIIFTAG K SISVEVLETLFKKIIKEVITDEQ, as shown in SEQ ID NO.1.

[0056] The gene sequence encoding the methyltransferase mutant M.MedI-N377K is as follows:

[0057]

[0058] (2) Artificial synthesis of caSAM: Dissolve S-adenosylhomocysteine in ammonium bicarbonate solution, add iodoacetic acid for purification to obtain a caSAM solution with a concentration of 3 mmol / L.

[0059] (3) Prepare the carboxymethylation working solution: Dissolve Tris-HCl, sodium chloride, dithiothreitol, and ethylenediaminetetraacetic acid in deionized water with concentrations of 1 mol / L, 500 mmol / L, 10 mmol / L, and 10 mmol / L respectively, and adjust its pH to 7.5.

[0060] (4) Commercially synthesized 28-oligonucleotide-length DNA strands containing CG sites, and the sequences are shown in Table 1 below.

[0061] Table 1

[0062]

[0063] (5) Take 60 ng of each single-stranded DNA sample obtained in step (4) and anneal it in boiling water to form double strands.

[0064] (6) Add a certain amount of methyltransferase mutant M.MedI-N377K, 1 μL of carboxy-S-adenosyl-L-methionine (caSAM), and 1 μL of reaction buffer (pH 7.9) to the double-stranded DNA obtained in step (5), add deionized water to make the reaction system 10 μL, and react at 37°C for 2 hours. The dosage of methyltransferase mutant M.MedI-N377K is adjusted according to the specific activity of the carboxymethylated protein expressed during the experiment.

[0065] (7) Treat the carboxymethylated DNA obtained in step (6) at 95°C for 10 min for denaturation.

[0066] (8) Subsequently, perform enzymatic digestion on the single-stranded DNA in step (7). The enzymes used for enzymatic digestion are 1 U of DNase I, 100 U of nuclease S1, 30 U of alkaline phosphatase, and 1 U of phosphodiesterase. The enzymatic digestion reaction is carried out in Tris-HCl buffer at pH 7.0, the enzymatic digestion temperature is 37°C, and the reaction time is 5 hours. The products after enzymatic digestion are analyzed and detected using LC-MS / MS.

[0067] The results are shown in Figure 2 , and the mass spectra of the DNA double strands containing CG sites before and after treatment with M.MedI-N377K show that a signal of 5camC appears after treatment, indicating that M.MedI-N377K can effectively carboxymethylate and label the C at the CG site.

[0068] Example 2: Preparation and synthesis of deaminase A3A

[0069] The preparation and synthesis method of deaminase A3A is as follows:

[0070] The amino acid sequence of deaminase A3A used in the present invention refers to the National Center for Biotechnology Information (NCBI Gene ID: 200315).

[0071] Insert the gene sequence encoding the A3A protein into the pET-41a(+) plasmid vector, with a GST tag at the N-terminus of the protein and an 8xHis tag at the C-terminus. There is a human rhinovirus 3C protease (HRV 3C) cleavage site between the tag and the A3A protein, and the corresponding recombinant expression vector is constructed;

[0072] Transform the recombinant expression vector into Escherichia coli BL21(DE3)pLySs cells for protein expression of deaminase A3A.

[0073] After protein expression, purification is carried out using glutathione agarose beads. After digestion with HRV3C enzyme, the full-length A3A deaminase is obtained respectively.

[0074] The amino acid sequence of deaminase A3A is as follows:

[0075] MEASPASGPRHLMDPHIFTSNFNNGIGRHKTYLCYEVERLDNGTSVKMDQHRGFLHNQAKNLLCGFYGRHAELRFLDLVPSLQLDPAQIYRVTWFISWSPCFSWGCAGEVRAFLQENTHVRLRIFAARIYDYDPLYKEALQMLRDAGAQVSIMTYDEFKHCWDTFVDHQGCPFQPWDGLDEHSQALSGRLRAILQNQGN, as shown in SEQ ID NO.5.

[0076] The gene sequence encoding deaminase A3A is as follows:

[0077] atggaagccagcccagcatccgggccccgtcacttgatggatccacacatcttcacttccaactttaacaatggcattggacgtcataagacctacctgtgctacgaagtggagcgcctggacaatggcacctcggtcaagatggaccagcaccgtggcttttctccacaaccaggctaagaatcttctctgtggcttttacggccgccatgcggagctgcgcttcttggacctggttccttctttgcagttggacccggcccagatctaccgtgtcacttggttcatctcctggagcccctgcttctcctggggctgtgccggggaagtgcgtgcgttccttcaggagaacacacacgtgcgtctgcgtatcttcgctgcccgcatctatgattacgaccccctctataaggaggcactgcaaatgctgcgggatgctggggcccaagtctccatcatgacctacgatgaatttaagcactgctgggacacctttgtggaccaccagggatgtcccttccagccctgggatggactcgatgagcacagccaagccctgagtgggcgtctgcgggccattctccagaatcagggaaac, as shown in SEQ ID NO.6.

[0078] Example 3: Analysis of Standard Synthetic DNA

[0079] In this example, commercially synthesized DNA strands containing C and 5mC respectively, namely C-DNA and 5mC-DNA (DNA to be sequenced), were used as the detection objects. The methyltransferase mutant M.MedI-N377K prepared in Example 1 and the deaminase A3A in Example 2 were used in combination to perform single-base resolution sequencing analysis of 5mC on C-DNA and 5mC-DNA.

[0080] The analysis principle is as Figure 1As shown in the figure. The methyltransferase mutant M.MedI-N377K can specifically transfer the carboxymethyl group of carboxy-S-adenosyl-L-methionine (caSAM) to the C5 position of C in the CG site to generate 5-carboxymethylcytosine (5camC). 5-Methylcytosine (5mC) can be deaminated by the deaminase A3A to form thymine (T), which pairs with adenine (A) in the subsequent polymerase chain reaction (PCR). 5camC can resist the deamination of A3A and pair with guanine (G) in the subsequent PCR. Finally, in the Sanger sequencing results of the amplified products, 5mC is read as T and C is read as C. Therefore, by comparing the sequencing results of the original DNA to be sequenced and the sequencing results after the above treatment, find the sites that are read as CG in the original DNA to be sequenced and become TG sites, and the T site here is the 5mC site in the original DNA to be sequenced.

[0081] The specific steps are as follows:

[0082] (2) Prepare the deamination working solution: Dissolve 2-morpholinoethanesulfonic acid in deionized water at a concentration of 200 mM, add 1% (v / v) Triton X-100, and adjust its pH to 6.5.

[0083] (3) Commercially synthesize DNA strands containing C and 5mC respectively.

[0084] (4) Take 60 ng of each of the DNA obtained in step (3), add a certain amount of M.MedI-N377K (determine the most suitable carboxymethylation concentration according to the actual activity of the M.MedI-N377K used), 1 μL of caSAM, 1 μL of reaction buffer (pH 7.9), add deionized water to make the reaction system 10 μL, and react at 37 °C for 2 hours.

[0085] (5) Treat the carboxymethylated DNA obtained in step (4) at 95 °C for 10 min, then place it in an ice-water bath for 5 min to denature the double-stranded DNA into single-stranded DNA, add a certain amount of A3A (determine the most suitable deamination concentration according to the actual activity of the A3A used), 2 μL of reaction buffer (pH 6.5), add deionized water to make the reaction system 20 μL, and incubate in a 37 °C water bath for 4 hours. Then incubate in a 95 °C water bath for 10 minutes.

[0086] (6) Take the DNA (40 ng) after the above reaction for polymerase chain amplification reaction.

[0087] Reaction system: 5 μL of 10× amplification buffer, 2 μL each of 10 μmol / L forward and reverse primers, 40 ng of template DNA, 10 U of EpiMark® Hot Start Taq DNA polymerase, and deionized water was added to make the system volume 50 μL.

[0088] The annealing temperature for the amplification reaction was selected according to different regions. Amplification cycle time program: ① Denaturation at 95°C for 5 min; ② Denaturation at 95°C for 30 sec; ③ Annealing at 50 - 72°C for 30 sec (annealing temperature was adjusted according to the primer length); ④ Extension at 72°C for 30 sec; Steps ② - ④ were repeated 25 times; Extension at 72°C for 10 min, and stored at 4°C. The samples were subjected to Sanger sequencing.

[0089] The nucleotide sequences of the DNA strands used are shown in Table 2 below.

[0090] Table 2

[0091]

[0092] The results were as Figure 3 , after treatment with M.MedI - N377K and A3A, the C at the CG site in C - DNA was basically completely non - deaminated and read as C during sequencing, and the 5mC in 5mC - DNA was basically completely deaminated and read as T during sequencing. This result indicates that the combination of M.MedI - N377K and A3A can perform localization analysis on 5mC in DNA.

[0093] The above - described specific embodiments have described the implementation of the present invention in detail. However, the present invention is not limited to the specific details in the above - described embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple variations all fall within the protection scope of the present invention.

Claims

1. Application of a methyltransferase mutant and a deaminase in single-base resolution sequencing of DNA methylation, characterized in that: Carboxymethylation treatment is performed on the original DNA to be sequenced using the methyltransferase mutant M.MedI-N377K and carboxyl-S-adenosyl-L-methionine; Denaturation treatment to obtain single-stranded carboxymethylated DNA to be sequenced; The single-stranded carboxymethylated DNA to be sequenced is subjected to deamination treatment by using the deaminase, the methyltransferase mutant and the deaminase are inactivated, amplified, sequenced, and the sequencing result is compared with the sequencing result of the original DNA to be sequenced to obtain the site of 5-methylcytosine in the CG site of the original DNA to be sequenced; The amino acid sequence of the methyltransferase mutant M.MedI-N377K is shown in SEQ ID NO.

1.

2. The use of the methyltransferase mutant and deaminase according to claim 1 in single-base resolution sequencing of DNA methylation, characterized in that: The gene sequence encoding the methyltransferase mutant M.MedI-N377K is shown in SEQ ID NO.

2.

3. The use of the methyltransferase mutant and deaminase according to claim 1 in single-base resolution sequencing of DNA methylation, characterized in that: The amino acid sequence of the deaminase is shown in SEQ ID NO.

5.

4. The use of the methyltransferase mutant and deaminase in DNA methylation single-base resolution sequencing according to claim 3, characterized in that: The gene sequence encoding the deaminase is shown in SEQ ID NO.

6.

5. A methyltransferase mutant M.MedI-N377K, characterized in that: The amino acid sequence is shown in SEQ ID NO.

1.

6. The methyltransferase mutant M.MedI-N377K according to claim 5, characterized in that The gene sequence encoding the methyltransferase mutant M.MedI-N377K is shown in SEQ ID NO.

2.

7. A biomaterial, characterized in that: The biological material is any one of the following: (1) A nucleic acid molecule encoding the methyltransferase mutant M.MedI-N377K according to claim 5 or 6; (2) a recombinant vector comprising a gene fragment encoding the methyltransferase mutant M.MedI-N377K according to claim 5 or 6; (3) An engineered cell, wherein the engineered cell comprises the nucleic acid molecule or the recombinant vector.

8. A single-base resolution sequencing analysis method for 5-methylcytosine in the CG site of a DNA to be tested, characterized in that: The following steps are involved: Carboxymethylation treatment of the original DNA to be sequenced is performed using the methyltransferase mutant M.MedI-N377K and carboxy-S-adenosyl-L-methionine described in claim 5 or 6; Denaturation treatment to obtain single-stranded carboxymethylated DNA to be sequenced; Deamination treatment is performed on the single-stranded carboxymethylated DNA to be sequenced by using a deaminase, and the methyltransferase mutant and the deaminase are inactivated, followed by amplification and sequencing; The sequencing result is compared with the sequencing result of the original DNA to be sequenced to obtain the site of 5-methylcytosine in the CG site of the original DNA to be sequenced.

9. The single-base resolution sequencing analysis method for 5-methylcytosine in the CG site of the DNA to be tested according to claim 8, characterized in that: The concentration of carboxy-S-adenosyl-L-methionine is 200-400 μM.

10. A product for single-base resolution sequencing analysis of 5-methylcytosine in the CG site of a DNA to be tested, characterized in that: The product comprises the methyltransferase mutant M.MedI-N377K as described in claim 5 or 6, and deaminase.

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