Patent Application
20230151341
The present invention relates to the field of bioengineering technology, and in particular relates to a method for specifically modulating the methylation/demethylation status of genomic DNA and use thereof.
DNA methylation is one of the important modifications in epigenetic modulation and is called the “fifth base” in mammalian DNA except for the four bases of ATCG. As a covalent modification, DNA methylation plays an important role in normal differentiation and disease development and can be stably inherited in cell differentiation of higher eukaryotic organs, and it is found in zebrafish that DNA methylation can be passed on to the next generation through sperm. Under the influence of cell differentiation, disease and environment, the methylation status of DNA will change greatly.
Studies have shown that DNA methylation is closely related to the occurrence and development of tumors. Changes in DNA methylation status include hypermethylation and hypomethylation. In general, DNA hypermethylation in the promoter region of the gene has the effect of silencing gene expression, while hypomethylation activates gene expression. DNA analysis of different tumor cells showed that the probability of genetic mutations in cancerous cells was much lower than expected. In the transcriptome range, gene expression inhibition by promoter hypermethylation in colorectal cancer was detected, and it was found that up to 5% of known genes have abnormal promoter hypermethylation in tumor cells. Therefore, it can be speculated that DNA methylation changes may play a greater role in cell malignant transformation than genetic mutations.
Target-specific nucleic acid editing techniques, especially the specific editing of genomic DNA, have always been an important technical basis for gene therapy. With the deepening of epigenetics research, more and more studies have shown that the methylation of the genome is directly involved in transcriptional modulation and other modulation of the genome, while the promotor and enhancer regions of an active expression gene are usually hypomethylated. Therefore, a nucleotide editing technique capable of specific demethylation is very important for the transcriptional activation of silenced genes.
Currently, site-specific and region-specific demethylation processes have been reported. For example, genomic remodeling of germ cells is often accompanied by large-scale demethylation. In addition, 5mC can be oxidized by certain enzymes (such as Tet) to 5hmC, followed by NER or BER process to be finally demethylated. Xu Guoliang, et al., have reported and filed a patent application for demethylation by reagents such as Tet dioxygenase and thymidine DNA glycosylase in 2015, but this method has not been able to accurately edit a certain site, being an important bottleneck for use in gene therapy or experimental technology tools.
Certain members of the Apobec protein family have the ability to deaminate 5mC into T in single-stranded DNA. With such characteristics and the precise positioning ability of the CRISPR protein family, it has become possible to develop a system that can accurately edit methylation at a specific site in the genome.
In order to solve the above problems, the present invention provides a method for editing a target nucleic acid molecule, comprising the steps of:
The recombinant vector in the above steps may be a recombinant vector in which two vectors respectively encode the fusion protein (A) and the small guide RNA (sgRNA) (B), or a recombinant vector in which a recombinant vector encodes both the fusion protein (A) and the small guide RNA (sgRNA) (B).
In a preferred embodiment, the Apobec family protein at N-terminal of the fusion protein is selected from the group consisting of human Apobec3A or Apobec3H, or a protein having deamination activity with 95% or more homology to human Apobec3A or Apobec3H. More preferably, the Apobec protein is Apobec3H or Apobec3A.
In another preferred embodiment, the Cas9 family protein whose nuclease activity is inactivated at C-terminal of the fusion protein is the one obtained by mutating aspartic acid at position 10 and histidine at position 840 in the wild-type Cas9 protein to alanine and alanine, or the Cpf1 protein whose nuclease activity is inactivated at C-terminal of the fusion protein is the one obtained by mutating aspartic acid to alanine at position 908 in the wide-type Cpf1 protein.
In order to provide better spatial structural flexibility for the two protein domains of the fusion protein, a linker consisting of 3-14 motifs can be added between the two domains of the fusion protein. The motif is selected from (GGS). The longer the linker is, the higher the spatial flexibility of the protein is and the larger the editable target area is.
To facilitate expression and purification of the fusion protein, a purification tag sequence can also be included. A commonly used purification tag is 6xHis.
In a more preferred embodiment, the fusion protein is selected from any of the sequences of SEQ ID NOs. 201-207.
The present invention also provides a gene sequence encoding the above fusion protein sequence, which is preferably selected from the group consisting of SEQ ID NOs. 301-307.
The present invention also provides a recombinant vector comprising any of the above gene sequences, which may be a prokaryotic expression vector or a eukaryotic expression vector, including but not limited to a plasmid vector, a viral vector, and the like, for the purpose of subsequent experiments.
Another aspect of the invention provides a small guide RNA molecule. In a preferred embodiment, the small guide RNA is 60 to 80 bp in length. In another preferred embodiment, the complementary region of the small guide RNA to the target nucleic acid molecule is 18 to 25 bp in length, preferably 20 bp.
A method for editing a target nucleic acid molecule in vitro, comprising the steps of: (1) obtaining a recombinant vector encoding a fusion protein (A) and a small guide RNA (sgRNA) (B), wherein the fusion protein (A) comprises an Apobec family protein domain at N-terminal and a Cas9 family or a Cpf1 family protein domain whose nuclease activity is inactivated at C-terminal, and the small guide RNA has a complementary region to a target editing region of the target nucleic acid molecule, wherein the target editing region of the target nucleic acid molecule includes at least one methylated cytosine nucleotide;
The present invention also provides use of the method for editing a target nucleic acid molecule for specifically modulating genomic DNA methylation/demethylation status.
In the method for editing a target nucleic acid molecule according to the present invention, the target nucleic acid molecule contains at least one methylated cytosine nucleotide, the methylated cytidine nucleotide is associated with diseases such as cancer, genetic disorders, developmental errors and the like. The method for editing a target nucleic acid molecule can be used for the treatment of a disease associated with cytosine nucleotide methylation, including but not limited to diseases associated with abnormal cell differentiation.
In the present invention, the Apobec protein having deamination activity is guided to the methylated cytosine position of the target nucleic acid molecule to modify the methylated cytosine by the guidance of sgRNA and the specific binding function of the mutant Cas9 or Cpf1. Further, the methylated cytosine is removed by an in vivo DNA repair mechanism to achieve specific editing of the target nucleic acid molecule. The gene editing method of the present invention has high specificity and has no dependence on the upstream and downstream sequences of the target site, and thus has universal applicability. Moreover, the gene editing method of the present invention only edits the target, does not produce off-target effects, and does not introduce insertion or deletion mutations during editing, thus has low toxic side effects.
The Cas9 or Cpf1 protein is a double-stranded DNA nuclease that binds to a targeting sequence and cleaves double-stranded DNA under the action of a small guide RNA (sgRNA). The Cas9 protein whose nuclease activity is inactivated retains the activity of binding to the targeting sequence, but does not cleave the target site. In the present invention, the methylated cytosine in the targeted sequence region is deaminated by fusing the Cas9 or Cpf1 protein whose nuclease activity is inactivated with the Apobec protein having deamination activity and guiding the Apobec protein to the target sequence region of the target nucleic acid molecule by the mutated Cas9 protein or Cpf1 protein, so that the target Met-C becomes T under deamination and does not pair with G on the complementary chain to form a protrusion. The addition of an effective amount of TDG after termination of the reaction by high temperature (the main effect is to inactivate the fusion protein by high temperature, usually at a temperature of 90 to 95° C.) removes the mismatched T base, thereby forming a deletion at the editing target site of the substrate. The dsDNA then changes back to ssDNA and cleaves at the base deletion site by the combined action of an effective amount of EDTA, formamide and NaOH.
Based on the above experiments, the applicant has found that the fusion protein Apobec-dCas9 or Apobec-dCpf1 enables site-specifically editing of methylated cytosine site in the target sequence region, which does not rely on the upstream and downstream sequences of the methylated cytosine site, has universal applicability, does not cause off-target effects, and does not introduce other insertion or deletion mutations, so there are no other toxic side effects.
The details will be further described below by way of specific examples. However, it should be understood that the specific embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. The instruments, devices, reagents, methods and the like used in the present application are all instruments, devices, reagents and methods commonly used in the art unless otherwise specified.
Invitrogen was commissioned to synthesize 6His-NLS-Apobec3H-linker (GGS-GGS-GGS) dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGSGGS-GGS-GGS), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGS-GGS-GGSGGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS)-dCas9(Asp10Ala/His840A1a), 6HisNLS-Apobec3A-linker (GGS-GGS-GGS)-dCas9(Asp10Ala/Hi s840A1a) dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3 A-linker (GGS-GGS-GGS-GGSGGS-GGS-GGS)-dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3A-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS) dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGSGGS-GGS-GGS)-dCpf1(Asp908A1a) gene sequences, respectively SEQ ID NO. 301, NO. 302, NO. 303, NO. 304, NO. 305, NO. 306 and NO. 307, and a Nco I endonuclease site was introduced at the 5′ end of the gene fragment, and a Hind III endonuclease site was introduced at the 3′ end. The synthesized gene fragment and the pET28a (+) vector were respectively double digested with Nco I and Hind III, and the gene fragment and the vector fragment were ligated with T4 DNA ligase, and DH5a competent cells (Tiangen Biochemical Technology (Beijing) Co., Ltd.) were routinely transformed, and positive clones were selected according to kanamycin resistance, then the plasmids were extracted. The recombinant plasmid was identified by Nco I and Hind III double digestion and agarose gel electrophoresis. Meanwhile, Invitrogen was commissioned to sequence the recombinant plasmid, and the results of the sequencing were analyzed using BioEdit software. The results were identical to the designed sequence, indicating that the recombinant plasmid was successfully constructed.
The obtained positive clone plasmid was transformed into E. coli. BL21 (DE3) competent cells (Tiangen Biotechnology (Beijing) Co., Ltd.), and cultured overnight at 37° C. in LB medium containing 100 μg/mlkanamycin, and then transferred to 1 L of the same LB medium and cultured at 37° C. to OD=0.6 about. The medium was then cooled to 4° C. and induced to express for approximately 16 hours by the addition of 0.5 mM IPTG. The cells were collected by centrifugation at 4000 g and resuspended in lysis buffer (50 mM Tris pH=7.0, 1 M NaCl, 20% glycerol, 10 mM TCEP). The cells were lysed by ultrasonic method (6W output for 8 minutes, on for 20 seconds and off for 20 seconds), and the supernatant was separated by centrifugation at 25,000 g. The supernatant was incubated with Nickel resin (ThermoFisher) at 4° C. for 1 hour, then passed through a gravity column and washed with 40 ml of lysis buffer. The recombinant protein was eluted with a 285 mM lysis buffer, diluted to 0.1 M NaCl and concentrated to the appropriate concentration with a centrifuge tube. The quality and concentration of the recombinant protein were determined by SDS Page.
The recombinant protein sequences were SEQ ID NO. 201-207.
Based on the 34 dsDNA substrate sequences to be tested (SEQ ID NO. 39-54 and their complementary strands 55-70, 71-85 and their complementary strands 86-100, 101-104 and their complementary strands 105-108) and the pFYF320 vector sequence providing the sgRNA universal sequence, the sgRNA forward primer (SEQ ID NO. 2-17, 18-34, and 35-38) and the reverse primer (SEQ ID NO. 1) were respectively designed. The sgRNA was obtained from a linear DNA fragment containing the T7 promoter by TranscriptAid T7 High Yield Transcription Kit (ThermoFisher Scientific), using DpnI to remove the template DNA, and then purified using a MEGAclear Kit (ThermoFisher Scientific), and the mass was detected by UV absorption.
Invitrogen was commissioned to synthesize the forward and reverse oligonucleic acid strand sequences of the substrate sequence, wherein the 5′ end of the positive strand sequence was labeled with FAM fluorescent labeling. 2 OD single-stranded oligonucleic acid strands were separately dissolved in 500 μl of water, and an equal amount of the positive and negative chain solutions were mixed and allowed to stand for 5 minutes to obtain a double-stranded substrate (dsDNA).
Fifteen sequences as SEQ ID NO. 39-54 were used for the dCas9 fusion protein demethylation range test.
Fifteen sequences as SEQ ID NO. 71-85 were used for the dCas9 fusion protein demethylation range test.
Four sequences as SEQ ID NO. 101-104 were used to test the effect of the base located adjacent to upstream of the target site on activity.
The recombinant fusion protein obtained in Example 1 was separately mixed with the sgRNA obtained in Example 2 in a molar ratio of 1:1, and allowed to stand at room temperature for 5 minutes. The corresponding dsDNA substrate was added to a final concentration of 125 nM and reacted at 37° C. for 2 hours. After the obtained dsDNA was purified using EconoSpin micro spin column (Epoch Life Science), 1 unit of TDG (NEB) was added and reacted at 37° C. for 1 hour. After the reaction, 10 μl of formamide, 1 μl of 0.5 M EDTA, and 0.5 μl of 5 M NaOH were added, and the mixture was reacted at 95° C. for 5 minutes. The product was isolated on 10% TBE-urea gel.
The target DNA strand contained the target Met-C and the 3′ end was labeled with the fluorophore FAM. Under the action of the recombinant protein, Met-C was converted to T and thus could not be paired with G of the complementary strand. Under the action of TDG, the mismatched T was going to be excised, leaving a base deletion site. Under the action of formamide and NaOH, the double strand became a single strand and was further cleaved at the base deletion site, thereby forming a short strand labeled with a fluorescent group FAM. The long and short chain marked DNAs were separated in urea gel. If a long and a short band appeared on the gel, it indicated that the recombinant protein was active.
Invitrogen was commissioned to synthesize the forward and reverse oligonucleic acid strand sequences of the substrate sequence, wherein the 5′ end of the positive strand sequence was labeled with FAM fluorescent labeling. 2 OD single-stranded oligonucleic acid strands were separately dissolved in 500 μl of water, and an equal amount of the positive and negative chain solutions were mixed and allowed to stand for 5 minutes to obtain a double-stranded substrate (dsDNA). The recombinant fusion protein obtained in Example 1 was separately mixed with the sgRNA obtained in Example 2 in a molar ratio of 1:1, and allowed to stand at room temperature for 5 minutes. The corresponding dsDNA substrate was added to a final concentration of 125 nM and reacted at 37° C. for 2 hours. The reacted dsDNA was purified using EconoSpin micro spin column (Epoch Life Science) and submitted to BGI for pyrosequencing after sulfite treatment and amplication with designed primers.
(1) Cell culture
The HEK293 cell line or PC3 cell line was maintained in Dulbecco's Modified Eagle's Medium plus under an environment of 37° C. and 5% carbon dioxide.
(2) Construction of PX330 recombinant protein expression vector
Invitrogen was commissioned to synthesize 6His-NLS-Apobec3H-linker (GGS-GGS-GGS) dCas9(Asp10A1a/His840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGSGGS-GGS-GGS-GGS), 6His-NLS-Apobec3H-linker (GGS-GGS-GGS-GGSGGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS) dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3A-linker (GGS-GGS-GGS) dCas9(Asp10Ala/His840A1a)-dCas9(Asp10Ala/His840Ala), 6His-NLSApobec3A-linker (GGS-GGS-GGS-GGS-GGS-GGS-GGS) dCas9(Asp10Ala/His840A1a), 6His-NLS-Apobec3A-linker (GGS-GGS-GGSGGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS-GGS) dCas9(Asp10A1a/His840Ala), 6His-NLS-Apobec3H-linker (GGS-GGS-GGSGGS-GGS-GGS-GGS)-dCpf1(Asp908A1a) gene sequences, respectively SEQ ID NO. 301, NO. 302, NO. 303, NO. 304, NO. 305, NO. 306 and NO. 307, and a BamHI endonuclease site was introduced at the 5′ end of the gene fragment, and an AgeI endonuclease site was introduced at the 3′ end. The synthesized gene fragment and the pX330 vector (Addgene) were respectively double digested with BamHI and AgeI, and the gene fragment and the vector fragment were ligated with T4 DNA ligase. It was confirmed by sequencing that the recombinant vector was constructed correctly. The sgRNA vectors corresponding to the five intracellular experiments inserted the corresponding PCR products (obtained by PCR from forward primers 121, 123, 125, 127, 129 and reverse primers 1, 122, 124, 126, 128, 130) through MluI and SpeI double digestion.
(3) Transfection
A. One day before transfection, HEK293 cells or PC3 cells were inoculated in a medium that did not contain antibiotics, and the confluence of the cells at the time of transfection was 30-50%.
B. Preparation of transfection samples:
1 μl of 20 μM pX330 recombinant vector and 1.5 μl of cell transfection reagent Lipofectamine™ 2000 (Invitrogen) were diluted in 0.05 ml Opti-MEM (Invitogen), gently mixed and incubated for 5 minutes. The control group was a blank pX330 vector that did not clone any foreign gene.
The diluted pX330 recombinant vector and Lipofectamine™ 2000 (Invitrogen) were incubated at room temperature for 20 minutes to form a recombinant vector-Lipofectamine™ 2000 (Invitrogen) complex and a blank vector-Lipofectamine 2000 (Invitrogen) complex. The incubation time should not exceed 30 minutes, and a longer incubation time may reduce activity.
The vector-Lipofectamine™ 2000 complex was added to each well containing cells and medium, and the plate was gently shaken back and forth, and incubated at 37° C. in a CO2 incubator for 72 hours.
The transfected cells were harvested 3 days later and the genomic DNA was purified by Agencourt DNA dvance Genomic DNA Isolation Kit (Beckman Coulter). Sample preparation was carried out by the method of Example 5, and the obtained sample was subjected to pyrosequencing by BGI Shenzhen.
According to Example 2, the inventor synthesized 30 ssDNA (15 fusion proteins for dCas9, 15 fusion proteins for dCpf1) of 59 bases in length as reaction substrates, their complementary ssDNA, and corresponding sgRNA primers. The 5′ end of the reaction substrate ssDNA was modified by the fluorophore FAM with a methylated C (Met-C) in between, which is the target of editing. After the ssDNA formed a dsDNA substrate with its complementary strand, the Cas9 region of the recombinant protein bound to the corresponding region in the middle of the dsDNA under the guidance of the corresponding sgRNA, and melted about 20 bases in the region, that was, formed a single-stranded region in the middle of the dsDNA. The target Met-C was in this region and was named as substrate 4-20 based on its distance to the 5′-end double-stranded region (4-20 bases). When the recombinant protein bound to different sgRNAs and then interacted with the corresponding dsDNA substrates for a certain period of time, some of the target Met-C became T under deamination and did not pair with G on the complementary strand to form a protrusion. The addition of 1 Unit of TDG after termination of the reaction at high temperature removed the mismatched T base, resulting in a deletion at the editing target of the substrate. The dsDNA then changed back to ssDNA and was cleaved at the base deletion site by the combined action of EDTA (0.5 μl at a concentration of 0.5 M), formamide (10 μl) and NaOH (1 μl at 5 M). Since both the cleaved 5′-end short-chain ssDNA and the unacting ssDNA substrate had a specific FAM fluorophore label at the 5′ end, the relative ratio of the two could be accurately estimated, and the efficiency of the recombinant protein to change Met-C to T at this site could be inferred.
As shown in
It can also be seen from the results that A3H was slightly more active than A3A.
As can be seen from the results, the dCpf1 fusion protein with a linker of (GGS) 7 in length had similar activity, and the distance of the action range was 7-12 bases.
In the control group, the synthesized T was used as a positive control, and the wrong sgRNA and Cas-9 or Cpf1 without sgRNA were used as negative controls.
The control experiment was mainly to prove two problems: first, our method is feasible. One of the groups in which the formation of short-chain DNA were clearly seen was chosen, the same ssDNA substrate was synthesized but the Met-C therein was changed to T, that was, the function of the recombinant protein was artificially completed. The same operations were employed. As a result, the formation of short-chain DNA was also observed. It was proved that the short-chain DNA in the experimental results was actually produced by the action of the recombinant protein on the target DNA. Second, by continuing the next experimental procedure by allowing the recombinant protein not to bind to sgRNA or to bind to unpaired sgRNA, no short-chain DNA was produced, demonstrating that such editing was directed.
A recombinant protein (a linker of GGS*7, and Apobec protein of A3H) was used as a subject for the study on effect of the base located adjacent to upstream of the editing target site on demethylation activity.
Based on previous studies of the Apobec protein family, the base located adjacent to upstream of the editing target site has a direct effect on their activities. The substrate with Met-C at position 7 was selected and the previous base was changed to A, T, C and G, respectively. As shown in
When it had been demonstrated that the recombinant protein had an ideal ability to change Met-C to T outside the cell, it was desirable to further verify whether such activity remains in the cell, the intensity of the activity, and whether T is repaired into a normal C by the cell's own DNA repair mechanism after the reaction, thereby achieving the effect of site-specific demethylation. The applicant designed three sets of intracellular experiments, and the promoter regions of three different genes were selected for demethylation testing.
The first intracellular editing target was the two methylated C of the U.S. Pat. Nos. 17,741,472 and 17,741,474 loci on chromosome 11 in the HEK293 cell line, located in the promoter region of the gene MYOD1. As shown in
The second editing target was a methylated C of the 31138558 locus on chromosome 6 in the HEK293 cell line, located in the promoter region of the gene POUF1. As shown in
The third editing target was a methylated C of the 113875226 locus on chromosome 2 in the PC3 cell line, located in the promoter region of the gene IL1RN. As shown in
Recombinant vectors were separately constructed and transfected into cells using the method described in Example 6, and the editing results were evaluated by pyrosequencing.
Based on the sequencing results of the above experiments, the cases of base insertion and deletion occurring near the target site throughout the process were also counted. From the sequencing results, there was no phenomenon of insertion and deletion of bases around.
The nucleic acid sequences used in the examples are specifically shown in the following table.
The sequences of protein domains are as follows:
| Number | Date | Country | Kind |
|---|---|---|---|
| 201610550293.X | Jul 2016 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2017/088281 | 6/14/2017 | WO |
