WO2023108929A1 - 靶向dna去甲基化的方法、融合蛋白及其应用 - Google Patents

靶向dna去甲基化的方法、融合蛋白及其应用 Download PDF

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WO2023108929A1
WO2023108929A1 PCT/CN2022/081136 CN2022081136W WO2023108929A1 WO 2023108929 A1 WO2023108929 A1 WO 2023108929A1 CN 2022081136 W CN2022081136 W CN 2022081136W WO 2023108929 A1 WO2023108929 A1 WO 2023108929A1
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sequence
amino acid
truncated
fusion protein
dna
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余细勇
朱昱光
申翱
张灵敏
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广州医科大学
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Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for targeting DNA demethylation, a fusion protein and an application thereof.
  • DNA methylation is the most common form of DNA modification, usually by covalently attaching a methyl group to the 5th carbon position of cytosine, making cytosine (C) into 5-methylcytosine (5mC) .
  • DNA methylation leads to changes in chromatin structure, DNA and protein binding ability, etc., thereby affecting gene expression and genetic traits.
  • DNA methylation is reversible, usually methylation inhibits gene expression, while demethylation promotes gene expression. Thus, methylation and demethylation regulate gene expression without altering the DNA sequence and are among the most common types of epigenetic regulation.
  • Ras association domain family 1A Ras association domain family 1 A, RASSF1A is a typical tumor suppressor gene, and its encoded protein inhibits tumor formation by inhibiting Ras activation.
  • Loss of RASSF1A expression due to methylation of the promoter region of the RASSF1A gene in human tumors is a common event, and methylation inactivation of RASSF1A has been found in nearly 40 different cancers.
  • lung cancer one of the cancers with the highest morbidity and mortality rate in the world, hypermethylation of the RASSF1A promoter is found in 100% of small cell lung cancer cell lines (SCLC), 63% of non-small cell lung cancer cell lines (NSCLC), 30% It was detected in primary NSCLC, but not in normal lung epithelial cells. It can be seen that the hypermethylation of the promoters of tumor suppressor genes leading to the inactivation of tumor suppressor genes is one of the common characteristics of various tumors.
  • small molecule inhibitors of DNA methylation have been applied in the clinical treatment of various cancers.
  • small molecule inhibitors are non-selective demethylation, resulting in abnormal activation of a large number of genes that were originally silenced. Therefore, if a targeted DNA demethylation technology can be developed to specifically increase the expression of tumor suppressor genes by targeting the promoters of tumor suppressor genes, it will be an important development direction of cancer precision medicine.
  • targeted DNA demethylation can also be applied to the treatment of other diseases with abnormal DNA methylation, which will have extensive and huge clinical impact.
  • DNA methylation is widespread in prokaryotes and eukaryotes.
  • DNA demethylation in eukaryotes includes passive and active methods. Passive demethylation means that after DNA methyltransferase (DNMT) is inactivated, the newly replicated DNA cannot be methylated, and the original 5mC gradually decreases with cell division and replication.
  • DNMT DNA methyltransferase
  • animals and plants employ different strategies. Animals use dioxygenase TET (including TET1, TET2, TET3) to oxidize 5mC into a series of derivatives. On the one hand, these derivatives cannot be recognized by DNMT and cannot maintain the methylation state.
  • thymine DNA glycosylation An enzyme cleaves these oxidized derivatives, resulting in demethylation by base excision repair (BER).
  • TDG thymine DNA glycosylation An enzyme
  • BER base excision repair
  • DME DNA glycosidases
  • ROS1, DML2, and DML3 are used to directly recognize and excise 5mC, and then complete demethylation through BER.
  • Non-targeted methods mainly use small molecule inhibitors of DNA methylation, such as 5-Azacytidine (Azacitidine, Vidaza), 5-Aza-2'-deoxycytidine (5-aza-2'-deoxycytidine glycosides, decitabine), and siRNA gene silencing against DNMT, etc.
  • Targeting methods are mainly based on zinc finger proteins (ZNFs), transcription activator-like effectors (TALEs), clustered regularly interspaced short palindromic repeats (CRISPR) and other systems that have the ability to target DNA and express one or more genes.
  • ZNFs zinc finger proteins
  • TALEs transcription activator-like effectors
  • CRISPR clustered regularly interspaced short palindromic repeats
  • a demethylation effector so as to achieve the purpose of demethylation of a specific DNA sequence.
  • Currently used demethylation effectors include full-length TET1, truncated TET1 (TET1-CD), and full-length
  • the CRISPR system includes two key elements: single guide RNA (single guide RNA, sgRNA) and CRISPR-associated endonuclease (CRISPR-associated endonuclease, Cas).
  • single guide RNA single guide RNA
  • CRISPR-associated endonuclease Cas
  • Cas9 CRISPR-associated endonuclease
  • Cas9 can be derived from different bacteria.
  • the sgRNA consists of a skeleton sequence (scaffold) necessary for binding to Cas9 and a recognition sequence (targeting) that recognizes the target DNA. By changing the recognition sequence, different targets on the DNA or different target DNA can be selected.
  • sgRNA guides Cas9 to bind to a specific DNA target and cut DNA.
  • the nuclease cleavage activity of Cas9 depends on two domains, RuvC and HNH, which are responsible for cutting the two strands of DNA, respectively.
  • a nuclease-inactive Cas9 dead Cas9, dCas9
  • the specificity of CRISPR targeting DNA is determined by two aspects, one is the base pairing between the sgRNA recognition sequence and the DNA target sequence, and the other is the combination of Cas9 and a specific short DNA sequence.
  • This short DNA sequence is called the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Different sources of Cas9 recognize different PAM sequences.
  • SpCas9 contains 1368 amino acids, and the PAM sequence is 5'-NGG-3'.
  • the newly discovered Cas9 CjCas9 from Campylobacter jejuni contains 984 amino acids and is the smallest Cas9 found in nature.
  • the PAM sequence of CjCas9 is 5'-NNNVYRAC-3', 5'-NNNNACA-3 ' or 5'-NNNNYRAC-3'. Studies have shown that CjCas9 has the same excellent gene editing potential as SpCas9.
  • the fusion protein is too large.
  • the amino acid sequences of the most commonly used SpCas9 and demethylation effectors are relatively long, which makes the insertion fragments used for the fusion expression of the two on the expression vector too long, while the commonly used viral vectors for gene therapy (lentivirus, adeno-associated virus, etc.) ) has a size limit for foreign inserts, which affects viral vector packaging and in vivo applications, so it is difficult to achieve demethylation gene therapy based on viral delivery.
  • Mini-CRISD Miniature CjCas9-ROS1 Induced Specific Demethylation
  • Mini-CRISD is based on the CRISPR-CjCas9 system, which ensures good targeting; using the reduced CjCas9 protein and demethylation effector ROS1, it is convenient for demethylation gene delivery based on virus treatment; and the demethylation process does not produce 5mC series derivatives, and does not cause additional epigenetic signals.
  • the object of the present invention is to provide a targeted DNA demethylation method (Mini-CRISD) and the fusion protein used in the method.
  • the Mini-CRISD method of the present invention delivers engineered reduced ROS1 demethylation effectors to specific DNA sequences and/or specific genomic locations (such as CpG islands) through engineered narrowed CjCas9 to target delivery of demethylation activity , can achieve targeted demethylation of specific DNA.
  • the first aspect of the present invention provides a fusion protein, which includes CjCas9 (dCjCas9 ⁇ HNH) inactivated by truncated nuclease, and truncated ROS1 (ROS1 ⁇ N), with a linker
  • CjCas9 dCjCas9 ⁇ HNH
  • ROS1 ⁇ N truncated ROS1
  • the intermediate sequence connects the truncated CjCas9 and the truncated ROS1
  • the truncated CjCas9 (dCjCas9 ⁇ HNH) is: the CjCas9 protein is removed from its 481-640 amino acids, and the remaining Amino acid fragments connected by linkers and subjected to D8A point mutation (the 8th amino acid is mutated from D to A);
  • the truncated ROS1 (ROS1 ⁇ N) is: ROS1 protein is removed from its 1-509 positions Amino acids,
  • the second aspect of the present invention provides a nucleotide sequence encoding the above fusion protein, or a nucleotide sequence that is the above nucleotide sequence and has one or more nucleotide mutations but encodes the same amino acid.
  • the above-mentioned nucleotide sequence encodes the same amino acid sequence based on codon optimization or codon degeneracy.
  • an expression vector capable of expressing the above-mentioned fusion protein is provided.
  • the fourth aspect of the present invention provides a kit for targeting DNA demethylation, which includes an expression vector capable of expressing the above fusion protein, or includes the above fusion protein.
  • a targeted DNA demethylation method comprising the following steps:
  • the sgRNA sequence includes a backbone sequence combined with CjCas9 ⁇ HNH (truncated CjCas9) and a recognition sequence fully complementary to the target sequence on the target DNA;
  • the sixth aspect of the present invention provides the application of the above fusion protein or expression vector.
  • a fusion control protein which includes a truncated CjCas9 (dCjCas9 ⁇ HNH) and a truncated ROS1 (ROS1 ⁇ N) connected through an intermediate sequence including a linker.
  • the truncated CjCas9 is: the CjCas9 protein is removed from its 481-640 amino acids, the remaining amino acids are connected with a linker, and the amino acid of the D8A point mutation (the eighth amino acid is mutated from D to A) Fragment;
  • the truncated ROS1 (ROS1 ⁇ N) is: the ROS1 protein is removed from its 1-509 amino acid and 628-855 amino acid, and the remaining amino acid is connected with a linker.
  • the 971 amino acid fragment of the ROS1 protein The amino acid is not aspartic acid (Asp).
  • the targeted DNA demethylation method (Mini-CRISD) of the present invention is the first time that the truncated dCjCas9 is used as a tool for DNA targeting, and it is also the first time that the truncated ROS1 is applied to DNA demethylation, and it is the first time that the two After appropriate treatment, they are combined and applied to target DNA demethylation.
  • the advantages of Mini-CRISD mainly include: (1) The advantages of genetic regulation, the ROS1 glycosidase from plants does not produce 5mC derivatives during the demethylation process, avoiding the current use of TET1 and other animal-derived The problem of generating 5mC derivatives and introducing additional genetic signals in the method of demethylating effectors.
  • Mini-CRISD has a brand-new sequence design. Compared with the currently used CRISPR system, it has a smaller gene sequence and higher activity, making the expression vector smaller in size. And the difficult-to-achieve demethylation gene therapy based on viral delivery becomes possible. (3) The advantage of gene targeting, Mini-CRISD is based on the principle of CRISPR, compared with other targeting systems such as ZNFs and TALEs, the targeting effect is better, and the off-target rate is low.
  • Mini-CRISD can target gene promoter regions to achieve targeted demethylation of methylated genes and increase the expression of target genes, and the entire demethylation process will not cause additional epigenetic changes; the present invention
  • the Mini-CRISD provides a new method for in vivo and/or ex vivo and/or in vitro induced gene therapy for various diseases with abnormal DNA methylation phenomenon.
  • Fig. 1 is a schematic flow chart of the targeted DNA demethylation method of the present invention.
  • Figure 2 is a schematic diagram of the construction process of the Mini-CRISD vector plasmid.
  • Figure 3 is a schematic diagram of the vector plasmid of Mini-CRISD.
  • Figure 4 is a schematic diagram of the results of agarose gel electrophoresis after HpaII restriction endonuclease cleavage.
  • Fig. 5 is a schematic diagram of the demethylation testing process.
  • Fig. 6 is a schematic diagram of the demethylation effect on Mini-CRISD in Example 4.
  • Example 7 is a schematic diagram of the demethylation effect on Mini-CRISD in Example 5.
  • Fig. 8 is a schematic diagram of the relative position of the DNA target in the genome and the RASSF1A promoter in Example 6.
  • Fig. 9 is a schematic diagram of the demethylation effect on the promoter region of the tumor suppressor gene RASSF1A in Example 6.
  • Figure 10 is a schematic diagram of the demethylation effect on the promoter region of the tumor suppressor gene RASSF1A in Example 7.
  • Some embodiments of the present invention relate to a fusion protein comprising CjCas9 inactivated by a truncated nuclease (dCjCas9 ⁇ HNH) and a truncated ROS1 (ROS1 ⁇ N) via a flexible linker.
  • dCjCas9 ⁇ HNH truncated nuclease
  • ROS1 ⁇ N truncated ROS1
  • the middle sequence is connected, wherein, the truncated CjCas9 (dCjCas9 ⁇ HNH) is: the 481-640 amino acids of the CjCas9 protein are removed, the remaining amino acids are connected with a flexible linker, and the D8A point mutation is carried out (section 8 amino acids are mutated from D to A) amino acid fragment; the truncated ROS1 (ROS1 ⁇ N) is: ROS1 protein is removed from its 1-509 amino acids and 628-855 amino acids, and the remaining amino acids are replaced by flexible An amino acid fragment connected by a linker, and the amino acid at position 971 of the ROS1 protein is aspartic acid (Asp).
  • the truncated CjCas9 (dCjCas9 ⁇ HNH) is: the 481-640 amino acids of the CjCas9 protein are removed, the remaining amino acids are connected with a flexible linker, and the D8A point mutation is carried out (section 8 amino acids are mut
  • the amino acid composition of the truncated CjCas9 is as follows from the N-terminal to the C-terminal:
  • SEQ ID NO.1 or its sequence with one or more amino acid mutations, substitutions but unchanged biological activity, flexible linker, and SEQ ID NO.2 or its one or more amino acid mutations, substitutions but unchanged biological activity the sequence of.
  • the amino acid composition of the truncated ROS1 is as follows from the N-terminal to the C-terminal:
  • SEQ ID NO.3 or its sequence with one or more amino acid mutations, substitutions but unchanged biological activity, flexible linker, and SEQ ID NO.4 or its one or more amino acid mutations, substitutions but unchanged biological activity the sequence of.
  • the fusion protein of the present invention follows the sequence of CjCas9 ⁇ HNH-ROS1 ⁇ N, that is, the C-terminal of CjCas9 ⁇ HNH is fused with the N-terminal of ROS1 ⁇ N; but the fusion protein can also follow the sequence of ROS1 ⁇ N-CjCas9 ⁇ HNH, that is, the N-terminal of CjCas9 ⁇ HNH is fused with the C-terminal of ROS1 ⁇ N .
  • the fusion protein (CjCas9 ⁇ HNH-ROS1 ⁇ N) includes from the N-terminus to the C-terminus:
  • SEQ ID NO.1 or its sequence with one or more amino acid mutations, substitutions but unchanged biological activity, flexible linker, and SEQ ID NO.2 or its one or more amino acid mutations, substitutions but unchanged biological activity the sequence of;
  • SEQ ID NO.3 or its sequence with one or more amino acid mutations, substitutions but unchanged biological activity, flexible linker, and SEQ ID NO.4 or its one or more amino acid mutations, substitutions but unchanged biological activity the sequence of.
  • the truncated CjCas9 and the truncated ROS1 are connected by an intermediate sequence.
  • the intermediate sequence mainly includes an NLS nuclear localization signal peptide, a flexible linker, and an enzyme cutting site used in molecular cloning.
  • the intermediate sequence is shown as SPKKKRKVEASKLGGGGSGGGGSGGGGSVD.
  • the front end of the truncated CjCas9 of the fusion protein also has a preorder sequence, and the preorder sequence includes an NLS nuclear localization signal peptide and an enzyme cleavage site.
  • the preamble is SPKKKRKVEAS.
  • the linker which is a linker sequence used to connect polypeptides, can connect two polypeptides and naturally fold them into a desired structure. Usually, it is a short peptide with a certain hydrophobicity and certain stretchability.
  • the purpose of the invention is to separate the two fused amino acid sequences so as to alleviate the mutual interference between the two and maintain their respective activities and functions.
  • Such peptide linkers include, but are not limited to, for example, soft linkers composed of various amino acids: GGGGSGGGGSGGGGS, GSSGN, GGGSGG.
  • the nuclear localization signal provides the nuclear translocation of the protein connected to the NLS.
  • NLS nuclear localization signal
  • dCjCas9 ⁇ HNH and ROS1 ⁇ N will enter the nucleus after being connected to the NLS, improving the demethylation efficiency of DNA in the nucleus.
  • nuclear localization signals include, but are not limited to, SPKKKRKVEAS, GPKKKRKV.
  • a tag sequence can be added to the fusion protein.
  • the tag sequence is DYKDDDDK (FLAG tag) added to the preamble sequence.
  • nucleotide sequence encoding the above fusion protein or the above nucleotide sequence with one or more nucleotide mutations but encoding the same amino acid.
  • the above-mentioned nucleotide sequence encodes the same amino acid sequence based on codon optimization or codon degeneracy.
  • kits capable of expressing the above-mentioned fusion proteins.
  • the vector can be a plasmid, a virus (such as an adenoviral vector, a retroviral or lentiviral vector, or an adeno-associated viral vector) or other expression vectors known in the art.
  • a virus such as an adenoviral vector, a retroviral or lentiviral vector, or an adeno-associated viral vector
  • the vector further includes a DNA sequence for expressing sgRNA, and each sgRNA sequence includes a backbone sequence that binds to CjCas9 ⁇ HNH (truncated CjCas9) and a recognition sequence that recognizes the target DNA.
  • the nucleotides contained in the recognition sequence are correspondingly changed for different DNA targets and are completely complementary to the DNA target sequence.
  • the length of the recognition sequence is usually preferably 20, 21 or 22 nucleotides, but the length of the recognition sequence can be is from 10 to 35 nucleotides.
  • the backbone sequence is a fixed fragment of 73 nucleotides, as shown in SEQ ID NO.21.
  • the DNA sequence used to express the sgRNA backbone sequence on the vector is shown in SEQ ID NO.22.
  • DNA demethylation-targeting kit which includes an expression vector capable of expressing the above fusion protein, or includes the above fusion protein.
  • the kit further includes sgRNA sequences, and each sgRNA sequence includes a backbone sequence capable of binding to the truncated CjCas9 and a recognition sequence for recognizing target DNA.
  • the sgRNA sequence is obtained through expression in the above expression vector, that is, the expression vector also includes a DNA sequence for expressing the sgRNA.
  • the sgRNA sequence can also be expressed separately in another expression vector, and does not need to be expressed in the same vector used to express the fusion protein, that is, the kit also includes an expression vector capable of expressing the DNA sequence of the sgRNA.
  • the sgRNA sequence includes a backbone sequence combined with CjCas9 ⁇ HNH (truncated CjCas9) and a recognition sequence fully complementary to the target sequence on the target DNA;
  • obtaining the sgRNA includes inserting the DNA sequence expressing the sgRNA into the expression vector expressing the above fusion protein, or inserting the DNA expressing the sgRNA in another separate vector sequence; the sgRNA sequence can also be obtained synthetically.
  • inventions of the present invention relate to the application of the above-mentioned fusion protein or expression vector to induce targeted DNA demethylation in vivo and/or in vitro and/or in vitro.
  • in vivo and/or ex vivo use viral vectors (such as adeno-associated virus, lentivirus) or non-viral vectors (such as liposomes, nanomaterials) to deliver expression vectors or fusion proteins in vivo and/or ex vivo .
  • viral vectors such as adeno-associated virus, lentivirus
  • non-viral vectors such as liposomes, nanomaterials
  • viral vectors e.g., adeno-associated virus, lentivirus
  • non-viral vectors e.g., liposomes, nanomaterials
  • other delivery means e.g., electroporation, microinjection
  • the diseases caused by abnormal DNA methylation include tumors or cancers, or related diseases such as metabolic diseases.
  • a fusion control protein comprising truncated CjCas9 (dCjCas9 ⁇ HNH) and truncated ROS1 (ROS1 ⁇ N) connected by an intermediate sequence including a flexible linker
  • the truncated CjCas9 is as follows: the 481-640 amino acids of the CjCas9 protein are removed, the remaining amino acids are connected with a flexible linker, and a D8A point mutation is carried out (the 8th amino acid is mutated from D to A ) amino acid fragment; the truncated ROS1 (ROS1 ⁇ N) is: the ROS1 protein has its 1-509 amino acid and 628-855 amino acid removed, and the remaining amino acid is connected with a flexible linker.
  • the amino acid at position 971 of the ROS1 protein is not aspartic acid (Asp), that is, other amino acids other than aspartic acid.
  • the fusion control protein can be used for experimental control.
  • the amino acid at position 971 of the ROS1 protein is not aspartic acid (Asp), for example, aspartic acid is mutated into asparagine (Asn).
  • Mini-CRISD In order to realize Mini-CRISD, we engineered and truncated the CjCas9 and ROS1 proteins respectively, and performed fusion expression on this basis. Mini-CRISD greatly reduces the size of the fusion protein (that is, the size of the inserted sequence on the expression vector) while retaining the targeting and ROS1 demethylation functions of Cas9, which is convenient for gene delivery in vivo and in vitro, and at the same time Can retain the corresponding biological activity.
  • Mini-CRISD is first based on two proven findings: (1) After CjCas9 removes the HNH domain of amino acids 481-640 (called CjCas9 ⁇ HNH), the RuvC domain retains a single-chain in vitro experiments Cutting function (Yamada M, Watanabe Y, Gootenberg J S, et al.Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.Mol Cell,2017,65(6):1109-1121) (2) After removing the 1-509 and 628-855 amino acids of the N-terminal of the purified ROS1 in vitro, the remaining amino acid sequence (called ROS1 ⁇ N) retains the ability to demethylate in vitro experiments (Hong S, Hashimoto H, Kow Y W, et al.
  • the carboxy-terminal domain of ROS1 is essential for 5-methylcytosine DNA glycosylase activity. J Mol Biol, 2014, 426(22): 3703-3712).
  • dCjCas9 ⁇ HNH the CjCas9 endonuclease activity
  • ROS1 ⁇ N also has a demethylase in vivo activity
  • the fusion expressed protein has the ability to target DNA demethylation in vivo.
  • the advantages of the Mini-CRISD of the present invention mainly include: (1) the advantages of genetic regulation, the ROS1 glycosidase from plants does not produce 5mc derivatives during the demethylation process, avoiding the current Issues with the generation of 5mc derivatives and the introduction of additional genetic signals in methods using demethylated effectors from animals such as TET1.
  • Mini-CRISD has a brand-new sequence design. Compared with the currently used CRISPR system, it has a smaller gene sequence and higher activity, making the expression vector smaller in size. And the difficult-to-achieve demethylation gene therapy based on viral delivery becomes possible.
  • Mini-CRISD is based on the principle of CRISPR, compared with other targeting systems such as ZNFs and TALEs, the targeting effect is better, and the off-target rate is low.
  • Our targeted DNA demethylation technology is the first to use dCjCas9 as a tool for DNA targeting, and it is also the first to apply truncated ROS1 to targeted DNA demethylation.
  • the method for targeted DNA demethylation of the present invention comprises the following steps for construction (see Figure 1):
  • Mini-CRISD plasmid expressing the Mini-CRISD complex (dCjCas9 ⁇ HNH and ROS1 ⁇ N fusion expression protein and sgRNA).
  • the plasmid contains three key elements to achieve targeted DNA demethylation: (1) a DNA sequence that can express dCjCas9 ⁇ HNH.
  • dCjCas9 ⁇ HNH means that the CjCas9 protein removes its HNH domain (481-640 amino acids), the remaining amino acids are connected with a flexible linker (such as GGGSGG), and a D8A point mutation is performed.
  • a DNA sequence capable of expressing ROS1 ⁇ N is performed.
  • ROS1 ⁇ N refers to the removal of amino acids 1-509 and amino acids 628-855 of the ROS1 protein, and the remaining amino acids are connected with a flexible linker (such as GSSGN).
  • a DNA sequence used to express one or more sgRNAs each sgRNA sequence includes two parts, one part is a fixed scaffold sequence (scaffold) that binds to CjCas9 ⁇ HNH; the other part is the recognition that changes according to different DNA targets Targeting sequence (targeting), and this part can be replaced by a blank sequence (spacer); the blank sequence contains a restriction site (such as BsmbI, BbsI, etc.), which is convenient for replacing the blank sequence with a recognition sequence.
  • a restriction site such as BsmbI, BbsI, etc.
  • the plasmid also contains the following 4 auxiliary elements to achieve Mini-CRISD: (4) A nuclear localization signal peptide (NLS) is introduced at both ends of dCjCas9 ⁇ HNH, which is used to import the fusion expressed protein into the nucleus . (5) Use a flexible linker (such as GGGGSGGGGSGGGG) between dCjCas9 ⁇ HNH and ROS1 ⁇ N. (6) A promoter driving the expression of dCjCas9 ⁇ HNH-ROS1 ⁇ N sequence, such as CMV promoter, EF1 ⁇ promoter, etc.
  • NLS nuclear localization signal peptide
  • the target DNA sequence should contain the PAM sequence required by CjCas9, that is, the 3' end of the target sequence should be adjacent to 5'-NNNNACA-3', 5'-NNNNRYAC-3' or 5'-NNNVRYAC-3' (where N is any base of A/T/C/G, V is any base of A/G/C; R is any base of A/G; Y is any base of T/C).
  • N is any base of A/T/C/G
  • V is any base of A/G/C
  • R is any base of A/G
  • Y is any base of T/C.
  • Mini-CRISD plasmid into cells containing the target DNA.
  • introduce the constructed vector into cells by any effective introduction method such as lipofection and electroporation; for in vivo experiments, use any effective introduction method such as virus infection and liposome
  • the introduction method is to introduce all of the constructed vector, or a part of the vector containing its functional fragment, or the key elements of Mini-CRISD into the body.
  • Mini-CRISD is also suitable for situations where a single vector is not used, that is, the dCjCas9 ⁇ HNH-ROS1 ⁇ N fusion protein and sgRNA do not have to be constructed on the same vector as described in step 1.
  • fusion protein and sgRNA can be co-expressed using different vectors, or expressed separately and then mixed together to form a complex, or sgRNA can be obtained by chemical synthesis.
  • the targeted DNA demethylation provided by the present invention can be performed.
  • CjCas9 ⁇ HNH of the fusion protein binds to the sgRNA backbone sequence so that the fusion protein and sgRNA form a complex; then the complex binds to the target sequence of the target DNA through the sgRNA recognition sequence, and passes The demethylase activity possessed by ROS1 ⁇ N of the fusion protein realizes the demethylation of target DNA.
  • Mini-CRISD vector plasmid The construction of the Mini-CRISD vector plasmid, and the construction of the Mini-CRISD-dead (D971N active site mutation, dead means loss of demethylation ability) control plasmid (see Table 1 for the specific sequence).
  • the design idea of the plasmid is shown in Figure 2.
  • the design of the vector plasmid includes: (1) removing the HNH domain (481-640 amino acids) of CjCas9, and performing a D8A point mutation, so that CjCas9 loses the endonuclease activity and only retains the ability to target DNA (referred to as dCjCas9 ⁇ HNH). (2) The 1-509 and 628-855 amino acids of the ROS1 protein are removed, and the remaining amino acid sequence (called ROS1 ⁇ N) retains the demethylation activity. (3) dCjCas9 ⁇ HNH and ROS1 ⁇ N were expressed as a fusion protein (dCjCas9 ⁇ HNH-ROS1 ⁇ N) through a flexible linker. (4) The expression elements of two sgRNAs, each sgRNA sequence consists of a fixed skeleton sequence (scaffold) and a blank sequence (spacer) that can be removed by BsmbI or BbsI enzyme digestion.
  • nucleotides of the scaffold sequence are as follows, guuuuuagucccugaaaagggacuaaaauaaagaguuugcgggacucucugcgggguuacaauccccuaaaaccgc (SEQ ID NO.21).
  • the DNA sequence expressing the sgRNA backbone sequence is gttttagtccctgaaaagggactaaaataaagagtttgcgggactctgcggggttacaatcccctaaaaccgc (SEQ ID NO.22).
  • the Mini-CRISD expression plasmid carrying the sgRNA targeting the specific DNA is obtained.
  • Mini-CRISD is also suitable for situations where the above-mentioned carrier plasmid is not used, that is, the dCjCas9 ⁇ HNH-ROS1 ⁇ N fusion protein and sgRNA do not have to be constructed on the same plasmid as described in step 1.
  • the fusion protein and sgRNA can be co-expressed using different plasmids, or expressed in other non-plasmid forms such as mRNA, or other expression vectors known in the art such as viral vectors; for another example, the fusion protein can be expressed in vitro Purified, and mixed with sgRNA obtained by chemical synthesis to form a complex.
  • Mini-CRISD is not limited to the specific plasmids described in this example.
  • Mini-CRISD-dead control plasmid which makes ROS1 lose its ability to demethylate by introducing the D971N amino acid point mutation, as a control for Mini-CRISD in other examples below.
  • the specific construction method is as follows:
  • Point mutations were performed using the Mini-CRISD plasmid as a template.
  • Use Biyuntian QuickMutation Gene Site-Directed Mutagenesis Kit (Cat. No. D0206) to design two complementary primers according to the experimental requirements (see the table below for primer sequences).
  • ROS1-dead-D971N-F TTGGCATTCCCAGTGAACACAAACGTTGGAAG
  • ROS1-dead-D971N-R CTTCCAACGTTTGTGTTCACTGGGAATGCCAA
  • the point mutation PCR reaction system is as follows:
  • Example 2 Construction of fluorescent reporter plasmids with different degrees of methylation.
  • CpG methyltransferase M.SssI (NEB Company, Cat. No. M0226S) can be used to achieve different degrees of methylation of plasmid DNA.
  • HpaII enzyme digestion was performed on the methylated plasmid for identification. Since HpaII only cuts unmethylated plasmids, the degree of methylation of the plasmid can be evaluated according to the HpaII cutting situation. The detailed steps are as follows:
  • the pEGFP-N1 fluorescent reporter plasmid (Clontech Company) was methylated in vitro, and different degrees of methylation could be obtained by different incubation times of the plasmid with CpG methyltransferase M.SssI.
  • the in vitro methylation reaction system and conditions are as follows:
  • Example 3 Construction of a Mini-CRISD expression plasmid targeting the CMV promoter on the pEGFP-N1 fluorescent reporter plasmid. Select 4 recognition sequences on the CMV promoter that meet the sequence requirements of CjCas9PAM, see the table below:
  • Synthesize primers containing the above recognition sequences, synthesize 2 complementary primers for each recognition sequence, and the ends of the primers contain the sticky end sequence cut by BsmBI, and anneal the primers on the PCR machine to form a double chain with the BsmBI sticky end head.
  • Annealing method 100uM each of the two primers was dissolved in TE buffer, incubated at 95°C for 5 minutes, and then slowly cooled to room temperature.
  • the methylated fluorescent reporter plasmid was tested for demethylation using the Mini-CRISD method in A549 cells. The detailed steps are as follows.
  • A549 cells were evenly seeded in a 96-well plate at a density of 5000 cells/well, cultured in a constant temperature incubator at 37°C and 5% CO2 using Ham's F-12K medium (containing 10% fetal bovine serum).
  • Example 2 24 hours later, use lipofectamine 3000 to simultaneously transfect 50 ng of the 50% methylated fluorescent reporter plasmid obtained in Example 2 and the Mini-CRISD expression plasmid obtained in Example 3 (carrying P CMV -sgRNA3 , 4, 5, or 6).
  • the control group was simultaneously transfected with 50% methylated fluorescent reporter plasmid and Mini-CRISD vector (without sgRNA) plasmid, each with 50 ng.
  • FIG. 5 The testing idea of this embodiment is shown in FIG. 5 .
  • Genes whose promoters are methylated cannot be transcribed and protein expressed. Therefore, when the pEGFP-N1 fluorescent reporter plasmid is 100% methylated, the expression of GFP green fluorescent protein in cells cannot be observed; and when the pEGFP-N1 fluorescent reporter plasmid When the reporter plasmid is partially methylated, GFP green fluorescent protein is under-expressed in cells; and if Mini-CRISD can demethylate the promoter region, GFP green fluorescent protein can be re-expressed. Therefore, in this example, the fluorescence intensity of GFP is inversely linearly correlated with the methylation degree of the plasmid.
  • the fluorescence intensity of the 50% methylated fluorescent reporter plasmid in the cells is very low; after adding Mini-CRISD, Mini-CRISD deactivated the characteristics of the region under the guidance of sgRNA targeting the CMV promoter region. Methylation significantly increases the expression and fluorescence intensity of GFP fluorescent protein. In the control group without sgRNA, even the Mini-CRISD fusion protein did not increase the fluorescence intensity, confirming that the observed increase in GFP expression (ie, fluorescence intensity enhancement) was due to targeted DNA demethylation.
  • the methylated fluorescent reporter plasmid was tested for demethylation using the Mini-CRISD method in 293T cells. The detailed steps are as follows.
  • 293T cells were uniformly seeded in a 96-well plate at a density of 12,000 cells/well, and cultured in a constant temperature incubator at 37°C and 5% CO2 using DMEM high-glucose medium (containing 10% fetal bovine serum).
  • Example 2 24 hours later, use lipofectamine 3000 to simultaneously transfect 50 ng each of the 75% methylated fluorescent reporter plasmid obtained in Example 2 and the Mini-CRISD expression plasmid obtained in Example 3 (carrying P CMV -sgRNA3 , 4, 5, or 6).
  • the control group was simultaneously transfected with 50 ng each of 50% methylated fluorescent reporter plasmid and Mini-CRISD-dead (demethylation inactivation mutation) plasmid.
  • the tumor suppressor gene RASSF1A promoter region was targeted for demethylation by Mini-CRISD method, thereby increasing the expression level of RASSF1A in lung cancer cells. Include the following steps.
  • the above-mentioned recognition sequence was used to replace the blank sequence on the Mini-CRISD vector plasmid, and 6 Mini-CRISD expression plasmids targeting the human tumor suppressor gene RASSF1A promoter (carrying sgRNA from the promoter of the oncogene RASSF1A).
  • P RASSF1A -sgRNA-3 TTCCTTCCCTCCTTCGTCCCCT (SEQ ID NO. 15)
  • P RASSF1A -sgRNA-4 GCTTGCTAGCGCCCAAAGCCAG (SEQ ID NO. 16)
  • P RASSF1A -sgRNA-5 CTGAGCTCATTGAGCTGCGGGA (SEQ ID NO. 17)
  • P RASSF1A -sgRNA-7 TGCGACAAGGGATAAACCATTT (SEQ ID NO.19)
  • P RASSF1A -sgRNA-8 CCAGGGACCAGCTGCCGTGTGG (SEQ ID NO. 20)
  • A549 cells were evenly seeded in a 6-well plate at a density of 25,000 cells/well, using Ham's F-12K medium (containing 10% FBS); H1299 cells were evenly seeded in a 6-well plate at a density of 30,000 cells/well, using RPMI-1640 Medium (with 10% FBS). Cultured in a constant temperature incubator at 37°C, 5% CO 2 environment.
  • Mini-CRISD expression plasmid (carrying one of PRASSF1A -sgRNA3, 4, 5, 6, 7, 8) into A549 or H1299 cells. Replace with new medium after 6 hours.
  • the control group used Mini-CRISD-dead (demethylation inactivation mutation) plasmid 1ug.
  • decitabine a small molecule inhibitor of DNA methylation
  • Decitabine Sigma, product number A3656
  • DMSO DMSO
  • Add 2 ul of decitabine stock solution to make the final decitabine concentration 10uM, and replace with new decitabine-containing medium every 24 hours.
  • Mini-CRISD targeted and demethylated the promoter region of the tumor suppressor gene RASSF1A in A549 and H1299 cells, which significantly enhanced the RASSF1A gene in lung cancer cells and promoted the expression of the RASSF1A gene .
  • the effect of Mini-CRISD targeted demethylation was significantly better than that of non-targeted small molecule inhibitor (decitabine).
  • Example 7 Targeted increase of the expression level of the tumor suppressor gene RASSF1A by the Mini-CRISD method to promote the death of lung cancer cells.
  • Calcein-AM itself does not emit fluorescence. After entering the cell, it is cleaved by intracellular esterase to form an impermeable membrane. Calcein stays in the cell and emits strong green fluorescence; dead cells lack esterase activity, so Calcein-AM only labels living cells. cell. Propidium iodide (PI) cannot pass through the cell membrane of living cells, but can only pass through the dead cell membrane to reach the nucleus, and embed into the cell DNA double helix to produce red fluorescence, so PI only marks dead cells.
  • PI Propidium iodide
  • Mini-CRISD targeted the promoter region of the tumor suppressor gene RASSF1A and demethylated it, which promoted the expression of RASSF1A gene, resulting in the death of A549 cells.
  • Mini-CRISD There was a significant difference between the CRISD-dead group and the non-transfected group.

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Abstract

提供了一种靶向的DNA去甲基化方法(Mini-CRISD)和其中所用到的融合蛋白。Mini-CRISD方法通过工程化的缩小dCjCas9递送工程化的缩小ROS1去甲基化效应子至特定DNA序列和/或特定基因组位置(如CpG岛)来靶向递送去甲基化活性，可以对特定DNA实现靶向去甲基化。

Description

靶向DNA去甲基化的方法、融合蛋白及其应用 技术领域
本发明属于生物技术领域，具体是涉及一种靶向DNA去甲基化的方法、融合蛋白及其应用。
背景技术
DNA甲基化是最常见的一种DNA修饰形式，通常是在胞嘧啶5号碳位上共价连接一个甲基基团，使得胞嘧啶(C)转为5-甲基胞嘧啶(5mC)。DNA甲基化导致染色质结构、DNA与蛋白质结合能力等的改变，从而影响基因表达和遗传性状。DNA甲基化是可逆的，通常甲基化抑制基因表达，而去甲基化促进基因表达。因此，甲基化和去甲基化在不改变DNA序列的前提下调控了基因表达，是最常见的表观遗传调控类型之一。真核生物的DNA甲基化通常发生在基因组上CpG岛位置(CpG岛是指基因组富含CG双核苷酸的某些区段，主要位于基因的启动子和外显子区域)。正确的DNA甲基化与生长发育、生理状态等密切相关，错误的DNA甲基化会导致生长发育异常、促进癌症发生和发展。例如，Ras相关区域家族1A(Ras association domain family 1 A，RASSF1A)是一个典型的抑癌基因，其编码蛋白通过抑制Ras活化从而抑制肿瘤形成。人类肿瘤中因RASSF1A基因的启动子区域甲基化导致RASSF1A表达缺失是普遍事件，已在近40种不同癌症中发现RASSF1A的甲基化失活。而作为全世界发病率和死亡率最高的癌症之一的肺癌，RASSF1A启动子高甲基化在100％的小细胞肺癌细胞系(SCLC)、63％的非小细胞肺癌细胞系(NSCLC)、30％的原发性NSCLC中被检测到，在正常的肺上皮细胞中未有检出。由此可见，抑癌基因的启动子高甲基化导致抑癌基因失活是各类肿瘤的共同特征之一。基于这一现象，DNA甲基化小分子抑制剂已应用于多种癌症的临床治疗。但小分子抑制剂是非选择性去甲基化，导致大量原本沉默的基因异常激活。因此，如果能发展一种靶向的DNA去甲基化技术，通过靶向抑癌基因启动子从而特异性地提高抑癌基因表达，是癌症精准医疗的重要发展方向。同理，靶向的DNA去甲基化还可应用于存在DNA异常甲基化现象的其他疾病的治疗，将产生广泛而巨大的临床影响。
DNA甲基化在原核和真核生物中广泛存在。真核生物的DNA去甲基化包括被动和主动两种方式。被动去甲基化是指DNA甲基转移酶(DNMT)失活后，新复制的DNA无法甲基化，原有5mC随着细胞分裂和复制而逐渐减少。对于主动去甲基化，动物和植物采用了不同的策略。动物利用双加氧酶TET(包括TET1、TET2、TET3)将5mC氧化为一系列衍生物，一方面这些衍生物不能被DNMT识别从而无法维持甲基化状态，另一方面胸腺嘧啶DNA糖基化酶(TDG) 会切除这些氧化衍生物，通过碱基切除修复(BER)实现去甲基化。而植物中利用DNA糖苷酶DME(包括ROS1、DML2、DML3)直接识别并切除5mC，再通过BER完成去甲基化。
目前去甲基化技术可分为非靶向和靶向两类。非靶向的方法主要使用DNA甲基化小分子抑制剂，例如5-Azacytidine(阿扎胞苷、维达莎)、5-Aza-2'-deoxycytidine(5-氮杂-2'-脱氧胞苷、地西他滨)，以及针对DNMT的siRNA基因沉默等。靶向的方法主要在锌指蛋白(ZNFs)、转录激活因子样效应子(TALEs)、规律成簇间隔短回文重复(CRISPR)等具有靶向DNA能力的系统的基础上融合表达一个或多个去甲基化效应子，从而达到对特定DNA序列去甲基化的目的。目前使用的去甲基化效应子包括全长TET1、截短的TET1(TET1-CD)、全长的ROS1。
利用CRISPR的靶向DNA技术发展最为迅速。CRISPR系统包括两个关键元件：单向导RNA(single guide RNA，sgRNA)和CRISPR相关核酸内切酶(CRISPR-associated endonuclease，Cas)。自然界中Cas种类多样，Cas9是目前基因工程领域应用最多的一类，并且Cas9可以来源于不同的细菌。sgRNA由一段结合Cas9所必需的骨架序列(scaffold)和一段识别靶DNA的识别序列(targeting)组成，通过改变识别序列，可选择DNA上的不同靶点或不同的靶DNA。在体外或体内，sgRNA与Cas9形成复合体后，sgRNA引导Cas9结合到特定DNA靶点并切割DNA。Cas9的核酸酶切割活性取决于RuvC和HNH两个结构域，这两个结构域分别负责切割DNA两条链。通过工程化改造Cas9，可获得切割活性缺失、仍具有靶向能力的核酸酶失活的Cas9(dead Cas9，dCas9)。CRISPR靶向DNA的特异性由两方面决定的，一方面是sgRNA识别序列和DNA靶点序列之间的碱基配对，另一方面是Cas9和一个特定短DNA序列的结合。这个短DNA序列通常在DNA靶点序列的3'末端，被称为原型间隔区相邻基序(protospacer adjacent motif，PAM)。不同来源的Cas9识别不同的PAM序列。目前最常用的Cas9来自Streptococcus pyogenes(SpCas9)，SpCas9包含1368个氨基酸，PAM序列为5’-NGG-3’。相较于SpCas9，新发现的来自Campylobacter jejuni的Cas9(CjCas9)包含984个氨基酸，是自然界已发现Cas9中尺寸最小的，CjCas9的PAM序列为5’-NNNVYRAC-3’、5’-NNNNACA-3’或5’-NNNNYRAC-3’。研究表明CjCas9具有和SpCas9一样优秀的基因编辑潜力。
目前已开发的DNA去甲基化技术存在各自的不足。对于非靶向的去甲基化，由于非特异性降低了基因组整体的甲基化水平，大量基因会异常表达，严重制约其实际应用。对于靶向的去甲基化，其局限性主要有三方面：(1)靶向性不高。例如，相较于CRISPR，ZNFs和TALEs不但需要对每个DNA靶点进行专门设计，且存在与基因组的“脱靶”结合。(2)特异性不高。例如，目前最常用的去甲基化效应子是全长或截短TET1，但TET1去甲基化过程中产生的5mC 衍生物同样携带表观遗传信息，在去甲基过程中会造成额外的表观遗传效应。(3)融合蛋白过大。目前最常用的SpCas9和去甲基化效应子的氨基酸序列均较长，使得表达载体上用于二者融合表达的插入片段过长，而常用的基因治疗病毒载体(慢病毒、腺相关病毒等)对外源插入片段有大小限制，这就影响了病毒载体包装和体内应用，所以难以实现基于病毒递送的去甲基化基因治疗。
为了克服上述不足，我们开发了一种新的靶向的DNA去甲基化技术，即Miniature CjCas9-ROS1 Induced Specific Demethylation(缩小的CjCas9-ROS1诱导的特异性去甲基化，简称Mini-CRISD)。相较于现有技术，Mini-CRISD基于CRISPR-CjCas9系统，很好地保证了靶向性；使用缩小的CjCas9蛋白和去甲基化效应子ROS1，便于进行基于病毒递送的去甲基化基因治疗；并且去甲基化过程中不产生5mC系列衍生物，不造成额外的表观遗传信号。
发明内容
本发明的目的提供了一种靶向的DNA去甲基化方法(Mini-CRISD)和该方法中所用到的融合蛋白。
本发明所述Mini-CRISD方法通过工程化的缩小CjCas9递送工程化的缩小ROS1去甲基化效应子至特定DNA序列和/或特定基因组位置(如CpG岛)来靶向递送去甲基化活性，可以对特定DNA实现靶向去甲基化。
本发明的第一个方面，提供了一种融合蛋白，其包括由截短后的核酸酶失活的CjCas9(dCjCas9△HNH)，以及截短后的ROS1(ROS1△N)，具有连接子的中间序列，所述中间序列将截短后的CjCas9、截短后的ROS1连接，其中,所述截短后的CjCas9(dCjCas9△HNH)为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用连接子连接、并进行D8A点突变(第8个氨基酸由D突变为A)的氨基酸片段；所述截短后的ROS1(ROS1△N)为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用连接子连接的氨基酸片段，且ROS1蛋白的971位的氨基酸为天冬氨酸(Asp)。
本发明第二个方面，提供了编码上述融合蛋白的核苷酸序列，或为上述核苷酸序列且具有一个多个核苷酸突变，但编码相同氨基酸的核苷酸序列。例如上述核苷酸序列基础上由于密码子优化或密码子简并性的编码相同氨基酸的序列。
本发明第三个方面，提供能表达上述融合蛋白的表达载体。
本发明第四个方面，提供一种靶向DNA去甲基化的试剂盒，其包括能表达上述融合蛋白的表达载体，或者包括上述融合蛋白。
本发明的第五个方面，提供了一种靶向DNA去甲基化方法，包括以下步骤：
s1.构建表达上述融合蛋白的表达载体；
s2.获得sgRNA，所述sgRNA序列包括与CjCas9ΔHNH(截短后的CjCas9)结合的骨架序列和与靶DNA上的靶点序列完全互补的识别序列；
s3.将所述表达载体与所述sgRNA导入含有靶DNA的目标物，进行靶向去甲基化。
本发明的第六个方面，提供了上述融合蛋白或者表达载体的应用。
上述融合蛋白或者表达载体在体内和/或离体和/或体外诱导的靶向DNA去甲基化的应用。
上述融合蛋白或者表达载体在制备基于靶向DNA去甲基化进行癌症治疗的药物中的应用。
上述融合蛋白或者表达载体在制备DNA甲基化异常导致的疾病治疗的药物中的应用。
本发明的第七个方面，还提供了一种融合对照蛋白，其包括由截短后的CjCas9(dCjCas9△HNH)以及截短后的ROS1(ROS1△N)通过包括连接子的中间序列连接而成，其中,所述截短后的CjCas9为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用连接子连接、并进行D8A点突变(第8个氨基酸由D突变为A)的氨基酸片段；所述截短后的ROS1(ROS1△N)为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用连接子连接的氨基酸片段，且ROS1蛋白的971位的氨基酸不是天冬氨酸(Asp)。
本发明的靶向DNA去甲基化方法(Mini-CRISD)是首次将截短的dCjCas9作为DNA靶向的工具，也是首次将截短的ROS1应用于DNA去甲基化，更是首次将两者经过合适处理后结合在一起应用于靶向DNA去甲基化。相较现有技术，Mini-CRISD的优势主要有：(1)遗传调控的优势，来自于植物的ROS1糖苷酶在去甲基化过程中不产生5mC衍生物，避免了目前使用TET1等来自动物的去甲基化效应子的方法中产生5mC衍生物并引入的额外遗传信号的问题。(2)分子技术的优势，Mini-CRISD具有全新的序列设计，相对于目前使用的CRISPR系统具有较小的基因序列、较高的活性，使得表达载体具有更小的尺寸，使得原先因载体尺寸而难以实现的基于病毒递送的去甲基化基因治疗成为可能。(3)基因靶向性的优势，Mini-CRISD基于CRISPR原理，相较于其他靶向系统如ZNFs和TALEs的靶向效果更好，脱靶率低。Mini-CRISD可以靶向基因启动子区域，实现对甲基化基因的靶向去甲基化，提高目的基因的表达，并且整个去甲基化过程不会造成额外的表观遗传变化；本发明的所述Mini-CRISD为存在DNA异常甲基化现象的各类疾病的体内和/或离体和/或体外诱导的基因治疗提供了一种新方法。
附图说明
图1为本发明的靶向的DNA去甲基化方法的流程示意图。
图2为Mini-CRISD载体质粒的构建流程示意图。
图3为Mini-CRISD的载体质粒示意图。
图4为HpaII限制性内切酶切割后琼脂糖凝胶电泳结果示意图。
图5为去甲基化测试流程示意图。
图6为实施例4中对Mini-CRISD的去甲基化效果示意图。
图7为实施例5中对Mini-CRISD的去甲基化效果示意图。
图8为实施例6中DNA靶点在基因组和RASSF1A启动子上的相对位置示意图。
图9为实施例6中针对抑癌基因RASSF1A启动子区域去甲基化效果示意图。
图10为实施例7中针对抑癌基因RASSF1A启动子区域去甲基化效果示意图。
具体实施方式
为了便于理解本发明，下面将对本发明进行更全面的描述。本发明可以以许多不同的形式来实现，并不限于本文所描述的实施例。相反地，提供这些实施例的目的是使对本发明公开内容的理解更加透彻全面。
下列实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件，或按照制造厂商所建议的条件。实施例中所用到的各种常用化学试剂，均为市售产品。
除非另有定义，本发明所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不用于限制本发明。本发明所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
本发明的一些实施例中涉及一种融合蛋白，该融合蛋白包括由截短后的核酸酶失活的CjCas9(dCjCas9△HNH)以及截短后的ROS1(ROS1△N)通过包括柔性连接子的中间序列连接而成，其中,所述截短后的CjCas9(dCjCas9△HNH)为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用柔性的连接子连接、并进行D8A点突变(第8个氨基酸由D突变为A)的氨基酸片段；所述截短后的ROS1(ROS1△N)为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用柔性的连接子连接的氨基酸片段，且ROS1蛋白的971位的氨基酸为天冬氨酸(Asp)。
在其中一些实施例中，所述截短后的CjCas9的氨基酸组成从N端到C端依次为：
SEQ ID NO.1或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、柔性连接子、和SEQ ID NO.2或其经过一个或多个氨基酸突变、取代但生物活性不变的序列。
在其中一些实施例中，所述截短后的ROS1的氨基酸组成从N端到C端依次为:
SEQ ID NO.3或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、柔性连接子、和SEQ ID NO.4或其经过一个或多个氨基酸突变、取代但生物活性不变的 序列。
本发明所述融合蛋白按照CjCas9ΔHNH-ROS1ΔN的顺序，即CjCas9ΔHNH的C端与ROS1ΔN的N端融合；但所述融合蛋白也可以按照ROS1ΔN-CjCas9ΔHNH的顺序，即CjCas9ΔHNH的N端与ROS1ΔN的C端融合。
在其中一些优选的实施例中，所述融合蛋白(CjCas9ΔHNH-ROS1ΔN)从N端到C端的包括：
SEQ ID NO.1或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、柔性连接子、和SEQ ID NO.2或其经过一个或多个氨基酸突变、取代但生物活性不变的序列；
中间序列；
SEQ ID NO.3或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、柔性连接子、和SEQ ID NO.4或其经过一个或多个氨基酸突变、取代但生物活性不变的序列。
在其中一些实施例中，所述截短后的CjCas9和截短后的ROS1之间由中间序列连接。所述中间序列主要包括NLS核定位信号肽、柔性的连接子、和分子克隆中使用的酶切位点。
在本发明的一些实施例中，所述中间序列如SPKKKRKVEASKLGGGGSGGGGSGGGGSVD所示。
在其中一些实施例中，所述融合蛋白的截短后的CjCas9的前端还具有前序序列，所述前序序列包括NLS核定位信号肽和酶切位点。例如，该前序序列为SPKKKRKVEAS。
所述连接子，其为用于可连接多肽的接头序列，其能够连接两个多肽并将其自然折叠成所期望结构，通常其是具有一段有疏水性和一定伸展性的短肽，在本发明中的目的是可将融合的两段氨基酸序列分开，以缓解二者相互干扰作用，并且保持其各自的活性和功能。此类肽接头包括但不限于例如各种以下氨基酸组成的软性连接子：GGGGSGGGGSGGGGS，GSSGN，GGGSGG。
核定位信号(NLS)提供NLS与其连接的蛋白质的核转运，例如在本发明中，dCjCas9△HNH和ROS1△N与NLS相连后将进入细胞核，提高在细胞核中对DNA的去甲基化效率。此类核定位信号包括但不限于，SPKKKRKVEAS，GPKKKRKV。为了便于在蛋白表达和定位研究中检测融合蛋白的表达，可以在融合蛋白中加入标签序列，在本发明的一些实施例中，该标签序列为在前序序列中加入了DYKDDDDK(FLAG标签)。
本发明的另一些实施例中涉及编码上述融合蛋白的核苷酸序列，或为上述核苷酸序列且具有一个多个核苷酸突变，但编码相同氨基酸的核苷酸序列。例如上述核苷酸序列基础上由于密码子优化或密码子简并性的编码相同氨基酸的序列。
本发明的另一些实施例中涉及能表达上述融合蛋白的表达载体(Mini-CRISD质粒)。
所述载体可以是质粒、病毒(如腺病毒载体、逆转录病毒或慢病毒载体，或腺相关病毒载 体)或本领域所知的其他表达载体。
在其中一些实施例中，所述载体还包括用于表达sgRNA的DNA序列，每个sgRNA序列包括与CjCas9ΔHNH(截短后的CjCas9)结合的骨架序列和识别靶DNA的识别序列。
所述识别序列所含的核苷酸针对不同DNA靶点相应改变、并与DNA靶点序列完全互补，识别序列的长度通常优选是20、21或22个核苷酸，但识别序列的长度可以是从10到35个核苷酸。
所述骨架序列是固定不变的73个核苷酸的片段，其如SEQ ID NO.21所示。相应地，载体上用于表达sgRNA骨架序列的DNA序列如SEQ ID NO.22所示。
本发明的另一些实施例中涉及一种靶向DNA去甲基化的试剂盒，其包括能表达上述融合蛋白的表达载体，或者包括上述融合蛋白。
在其中一些实施例中，所述试剂盒还包括sgRNA序列，每个sgRNA序列包括能与所述截短后的CjCas9结合的骨架序列和识别靶DNA的识别序列。
所述sgRNA序列在上述表达载体中表达获得，即所述表达载体还包括用于表达sgRNA的DNA序列。
所述sgRNA序列也可以单独在另一个表达载体中表达获得，不需要在上述表达融合蛋白的同一载体中表达，即所述试剂盒还包括能表达sgRNA的DNA序列的表达载体。
本发明的另一些实施例中涉及一种靶向DNA去甲基化方法，包括以下步骤：
s1.构建表达上述融合蛋白的表达载体；
s2.获得sgRNA，所述sgRNA序列包括与CjCas9ΔHNH(截短后的CjCas9)结合的骨架序列和与靶DNA上的靶点序列完全互补的识别序列；
s3.将所述表达载体与所述sgRNA导入含有靶DNA的目标物，进行靶向去甲基化。
上述步骤s2中，获得所述sgRNA，包括将在表达上述融合蛋白的表达载体中插入有表达所述sgRNA的DNA序列，也可以是在另一个单独的载体中插入表达所述所述sgRNA的DNA序列；也可以是合成获得所述sgRNA序列。
本发明的另一些实施例中涉及上述融合蛋白或者表达载体在体内和/或离体和/或体外诱导的靶向DNA去甲基化的应用。
具体的，体内和/或离体：使用病毒载体(例如腺相关病毒、慢病毒)或非病毒载体(例如脂质体、纳米材料)，将表达载体或者融合蛋白递送到体内和/或离体。
体外：使用病毒载体(例如腺相关病毒、慢病毒)或非病毒载体(例如脂质体、纳米材料)或其他递送手段(例如电穿孔、显微注射)，将表达载体或者融合蛋白递送到细胞中。
上述融合蛋白或者表达载体在制备基于靶向DNA去甲基化进行癌症治疗的药物中的应用。
上述融合蛋白或者表达载体在制备DNA甲基化异常导致的疾病治疗的药物中的应用。
所述DNA甲基化异常导致的疾病包括肿瘤或者癌症，或者代谢性疾病等相关疾病。
本发明的一些实施例中，还涉及到一种融合对照蛋白，其包括由截短后的CjCas9(dCjCas9△HNH)以及截短后的ROS1(ROS1△N)通过包括柔性连接子的中间序列连接而成，其中,所述截短后的CjCas9为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用柔性的连接子连接、并进行D8A点突变(第8个氨基酸由D突变为A)的氨基酸片段；所述截短后的ROS1(ROS1△N)为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用柔性的连接子连接的氨基酸片段，且ROS1蛋白的971位的氨基酸不是天冬氨酸(Asp)，即可为非天冬氨酸的其他氨基酸。
所述融合融合对照蛋白，可以用于实验对照，ROS1蛋白的971位的氨基酸不是天冬氨酸(Asp)，例如为天冬氨酸突变为天门冬酰胺(Asn)。
以下通过具体的实施例对本发明做进一步的阐述，但不用于限制本发明的保护范围。
实施例1
为了实现Mini-CRISD，我们分别对CjCas9和ROS1蛋白进行工程化改造截短、并在此基础上进行融合表达。Mini-CRISD在保留Cas9的靶向性、ROS1去甲基化功能的同时，极大地缩小了融合蛋白的尺寸(亦即表达载体上插入序列的大小)，便于体内和体外的基因递送，同时又能保留相应的生物活性。Mini-CRISD首先基于已被证实的2个发现：(1)CjCas9在移除481-640位氨基酸的HNH结构域后(被称为CjCas9△HNH)，RuvC结构域在体外实验中保留了单链切割功能(Yamada M,Watanabe Y,Gootenberg J S,et al.Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems.Mol Cell,2017,65(6):1109-1121)；(2)体外纯化的ROS1在移除蛋白N端的1-509位和628-855位氨基酸后，剩余的氨基酸序列(被称作ROS1ΔN)在体外实验中保留了去甲基化的能力(Hong S,Hashimoto H,Kow Y W,et al.The carboxy-terminal domain of ROS1 is essential for 5-methylcytosine DNA glycosylase activity.J Mol Biol,2014,426(22):3703-3712)。在此基础上，我们进一步通过D8A点突变，彻底失活CjCas9内切酶活性，仅保留其靶向DNA能力(被称作dCjCas9ΔHNH)；同时我们也证实了ROS1ΔN在体内同样具有去甲基化酶活性；再通过将dCjCas9ΔHNH与ROS1ΔN融合表达，并配合相应的sgRNA，使得融合表达蛋白具备体内靶向的DNA去甲基化能力。并且我们创造性地使用截短的dCjCas9ΔHNH和截短的ROS1ΔN蛋白，这极大地缩小了融合蛋白的尺寸，使表达载体上插入序列极大地缩小，从而实现体内和体外的基因递送，同时又能保留相应的生物活性。
相较于现有技术，本发明所述Mini-CRISD的优势主要有：(1)遗传调控的优势，来自于植物的ROS1糖苷酶在去甲基化过程中不产生5mc衍生物，避免了目前使用TET1等来自动物的去 甲基化效应子的方法中产生5mc衍生物并引入的额外遗传信号的问题。(2)分子技术的优势，Mini-CRISD具有全新的序列设计，相对于目前使用的CRISPR系统具有较小的基因序列、较高的活性，使得表达载体具有更小的尺寸，使得原先因载体尺寸而难以实现的基于病毒递送的去甲基化基因治疗成为可能。(3)基因靶向性的优势，Mini-CRISD基于CRISPR原理，相较于其他靶向系统如ZNFs和TALEs的靶向效果更好，脱靶率低。我们的靶向DNA去甲基化技术是首次将dCjCas9作为DNA靶向的工具，也是首次将截短的ROS1应用于靶向DNA去甲基化。
本发明所述靶向DNA去甲基化的方法，包含以下步骤进行构建(参见图1)：
1.构建表达Mini-CRISD复合物(dCjCas9ΔHNH与ROS1ΔN融合表达蛋白及sgRNA)的表达质粒载体(简称为Mini-CRISD质粒)。该质粒包含实现靶向的DNA去甲基化的3个关键元件：(1)一段DNA序列，可以表达dCjCas9ΔHNH。dCjCas9ΔHNH是指CjCas9蛋白移除其HNH结构域(481-640位氨基酸)、剩余氨基酸用柔性的连接子(如GGGSGG)连接、并进行D8A点突变。(2)一段DNA序列，可以表达ROS1ΔN。ROS1ΔN是指ROS1蛋白移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用柔性的连接子(如GSSGN)连接。(3)一段DNA序列，用于表达一条或多条sgRNA，每个sgRNA序列包括2部分，一部分是固定的与CjCas9ΔHNH结合的骨架序列(scaffold)；另一部分是根据不同DNA靶点而改变的识别序列(targeting)，并且该部分可以由一段空白序列(spacer)代替；空白序列上包含酶切位点(例如BsmbI、BbsI等)，便于将空白序列替换为识别序列。除上述3个关键元件外，该质粒还包含以下4个辅助元件以实现Mini-CRISD：(4)在dCjCas9ΔHNH的两端引入核定位信号肽(NLS)，用于将融合表达的蛋白导入细胞核中。(5)在dCjCas9ΔHNH和ROS1ΔN之间用柔性的连接子(如GGGGSGGGGSGGGG)连接。(6)驱动dCjCas9ΔHNH-ROS1ΔN序列表达的启动子，如CMV启动子、EF1α启动子等。(7)驱动sgRNA序列转录的启动子，如U6启动子、H1启动子等。此外，靶DNA序列上应含有CjCas9所需的PAM序列，即靶点序列的3’端应紧邻5’-NNNNACA-3’、5’-NNNNRYAC-3’或5’-NNNVRYAC-3’(其中N为A/T/C/G任一碱基，V为A/G/C任一碱基；R为A/G任一碱基；Y为T/C任一碱基)。根据DNA靶点序列设计相应的识别序列并替换掉Mini-CRISD质粒上的空白序列，获得针对特定DNA靶点的Mini-CRISD质粒(携带靶向特定DNA的sgRNA)。
2.将Mini-CRISD质粒导入含有靶DNA的细胞中。对于体外/离体实验，通过脂质体转染、电穿孔等任何一种有效的导入方法将所构建的载体导入细胞；对于体内实验，通过病毒感染、脂质体包裹等任何一种有效的导入方法，将所构建的载体全部、或含有其功能片段的部分载体、或Mini-CRISD的关键元件导入体内。通过dCjCas9ΔHNH-ROS1ΔN融合蛋白和sgRNA组装为复合物后，将靶向特定DNA序列并实现去甲基化这一目的。
需要强调的是，Mini-CRISD也适用于不使用单个载体的情形，即dCjCas9ΔHNH-ROS1ΔN融合蛋白和sgRNA不是必须如步骤1所述构建在同一载体上。例如，融合蛋白和sgRNA可以使用不同的载体进行共表达，或单独表达后再混合在一起形成复合物，或sgRNA通过化学合成的方式获得。只要通过适当的手段获得了dCjCas9ΔHNH-ROS1ΔN融合蛋白和sgRNA，就可以进行本发明提供的靶向DNA去甲基化。
靶向DNA去甲基化时，所述融合蛋白的CjCas9ΔHNH与sgRNA骨架序列结合从而使融合蛋白与sgRNA形成复合物；然后该复合物通过sgRNA识别序列结合到靶DNA的靶点序列上，并通过融合蛋白的ROS1ΔN所具有的去甲基化酶活性实现对靶DNA的去甲基化。
Figure PCTCN2022081136-appb-000001
Figure PCTCN2022081136-appb-000002
Figure PCTCN2022081136-appb-000003
Figure PCTCN2022081136-appb-000004
Figure PCTCN2022081136-appb-000005
Figure PCTCN2022081136-appb-000006
Figure PCTCN2022081136-appb-000007
Figure PCTCN2022081136-appb-000008
Mini-CRISD载体质粒的构建，以及Mini-CRISD-dead(D971N活性位点突变，dead意为丧失了去甲基化能力)对照质粒的构建(具体序列见表1)。质粒的设计思路如图2所示。
本发明质粒构建的详细步骤如下：
1.通过全基因合成的方式构建一个包含上述技术方案步骤1中所述的Mini-CRISD各关键和辅助元件的载体质粒(如图3所示)，该合成由南京擎科生物科技有限公司完成。
该载体质粒的设计包括：(1)移除CjCas9的HNH结构域(481-640位氨基酸)、并进行D8A点突变，从而使CjCas9失去内切酶活性，仅保留靶向DNA能力(被称作dCjCas9ΔHNH)。(2)移除ROS1蛋白的1-509位、628-855位氨基酸，剩余氨基酸序列(被称作ROS1ΔN)保留去甲基化活性。(3)dCjCas9ΔHNH与ROS1ΔN通过柔性连接子表达为一个融合蛋白(dCjCas9ΔHNH-ROS1ΔN)。(4)两个sgRNA的表达元件，每个sgRNA序列由固定的骨架序列(scaffold)和可通过BsmbI或BbsI酶切移除的空白序列(spacer)组成。
骨架序列(scaffold)核苷酸如下所示，guuuuagucccugaaaagggacuaaaauaaagaguuugcgggacucugcgggguuacaauccccuaaaaccgc(SEQ ID NO.21)。
相应地，表达sgRNA骨架序列的DNA序列为gttttagtccctgaaaagggactaaaataaagagtttgcgggactctgcggggttacaatcccctaaaaccgc(SEQ ID NO.22)。
当通过适当的分子克隆手段，将载体质粒上的空白序列替换为针对特定DNA靶点的识别序列(targeting)后，即获得携带靶向特定DNA的sgRNA的Mini-CRISD表达质粒。
需要强调的是，Mini-CRISD也适用于不使用上述载体质粒的情形，即dCjCas9ΔHNH-ROS1ΔN融合蛋白和sgRNA不是必须如步骤1中所述构建在同一质粒上。例如，融合蛋白和sgRNA可以使用不同的质粒进行共表达，或者通过mRNA等其他非质粒的形式进行表达，或者使用病毒载体等本领域所知的其他表达载体；再例如，融合蛋白可以通过体外表达纯化的方式获得， 并与通过化学合成的方式获得的sgRNA混合在一起形成复合物。只要通过适当手段获得了dCjCas9ΔHNH-ROS1ΔN融合蛋白和sgRNA，就可以进行本发明提供的靶向DNA去甲基化。因此Mini-CRISD不限于本实施例中所描述的特定质粒。
2.构建Mini-CRISD-dead对照质粒，该质粒通过引入D971N氨基酸点突变使得ROS1失去了去甲基化能力，作为后文其他实施例中Mini-CRISD的对照。具体构建方法如下：
以Mini-CRISD质粒为模板进行点突变。使用碧云天QuickMutation基因定点突变试剂盒(货号D0206)，按照实验要求设计两条互补引物(引物序列见下表)。
ROS1-dead-D971N-F TTGGCATTCCCAGTGAACACAAACGTTGGAAG(SEQ ID NO.9)
ROS1-dead-D971N-R CTTCCAACGTTTGTGTTCACTGGGAATGCCAA(SEQ ID NO.10)
点突变PCR反应体系如下：
试剂 最终浓度 体积
10X BeyoFusion缓冲液 1X 5μl
引物混合物(10μM) 0.4μM 2μl
dNTP mix(2.5mM) 0.25mM 5μl
Mini-CRISD质粒 200ng 1μl
BeyoFusion DNA聚合酶 1/50 1μl
加无核酶水后总体积 - 50μl
PCR的反应条件如下：
步骤 循环数 温度 时间
1 1 95℃ 3min
2 20 95℃ 30sec
    55℃ 30sec
    68℃ 60sec/kb
3 1 68℃ 15min
4 1 4℃ 长时间保持
PCR反应后，直接在PCR反应体系中加入1μl DpnI，混匀后37℃孵育5min。DpnI消化后，每100μl感受态细菌(擎科Trelief 5α Chemically Competent Cell货号：TSC-C01)中加入5-10μl经过DpnI消化后的产物，进行转化。对于得到的转化克隆子，挑取3-5个克隆送样测序去测序，测序结果显示获得了预期的点突变。
实施例2：构建不同程度甲基化的荧光报告质粒。
利用CpG甲基转移酶M.SssI(NEB公司，货号M0226S)不同的孵育时间，可以实现对质 粒DNA不同程度的甲基化。对甲基化后的质粒进行HpaII酶切鉴定，由于HpaII只切割未甲基化的质粒，根据HpaII切割的情况可以评估质粒的甲基化程度。详细步骤如下：
1.对pEGFP-N1荧光报告质粒(Clontech公司)进行体外甲基化，通过CpG甲基转移酶M.SssI对质粒不同的孵育时间可获得不同程度甲基化。体外甲基化反应体系和条件如下：
Figure PCTCN2022081136-appb-000009
2.对上述进行了体外甲基化反应的荧光报告质粒进行HpaII限制性内切酶切割。琼脂糖凝胶电泳后，根据HpaII切割和未切割的DNA含量确定甲基化的程度(图4)。
实施例3：构建靶向pEGFP-N1荧光报告质粒上CMV启动子的Mini-CRISD表达质粒。选取CMV启动子上符合CjCas9PAM序列要求的识别序列4条，见下表：
P CMV-sgRNA-3 TCAAACCGCTATCCACGCCCAT(SEQ ID NO.11)
P CMV-sgRNA-4 ATTGACGTCAATGGGAGTTTGT(SEQ ID NO.12)
P CMV-sgRNA-5 CATTGACGCAAATGGGCGGTAG(SEQ ID NO.13)
P CMV-sgRNA-6 CTCTGCTTATATAGACCTCCCA(SEQ ID NO.14)
1.对Mini-CRISD载体质粒进行BsmBI限制性内切酶切割。
2.琼脂糖凝胶电泳回收载体片段。
3.合成包含上述识别序列的引物，每个识别序列合成2条互补引物、且引物末端含有BsmBI切割后的粘端序列，在PCR仪上对引物进行退火反应以形成带BsmBI粘末端的双链接头。退火方式：2条引物各100uM溶于TE缓冲液中，95℃孵育5分钟后，缓慢降温至室温。
4.退火后的接头片段与酶切回收的Mini-CRISD载体质粒在T4连接酶16度条件下反应过夜。
5.转化并获得单克隆。测序显示表达质粒构建成功，即根据CMV启动子设计的识别序列替换掉了Mini-CRISD载体质粒上的空白序列，获得4个靶向CMV启动子的Mini-CRISD表达质粒(携带靶向CMV启动子的sgRNA)。
实施例4
在A549细胞中使用Mini-CRISD方法对甲基化的荧光报告质粒进行去甲基化测试。详细步骤如下。
1)A549细胞以5000个/孔的密度均匀接种于96孔板，使用Ham's F-12K培养基(含10％胎牛血清)，在37℃、5％CO 2的恒温培养箱中培养。
2)24小时后使用lipofectamine 3000向A549细胞同时转染从实施例2中获得的50％甲基化的荧光报告质粒与实施例3中获得的Mini-CRISD表达质粒各50ng(携带P CMV-sgRNA3、4、5、6中的一个)。对照组为同时转染50％甲基化的荧光报告质粒和Mini-CRISD载体(不含sgRNA)质粒各50ng。
3)48小时后去除培养基，PBS漂洗2遍后用4％多聚甲醛固定，15分钟后吸去4％多聚甲醛，PBS漂洗一遍，每孔加入30μL DAPI染色液，5分钟后吸去染色液，PBS漂洗2遍，每孔加入100μL PBS。
4)使用Thermo Scientific Cell insight CX7-LZR高内涵分析系统内置软件HCS Navigator Version6.6.1采集图像和数据处理。96孔板每孔选取25个视野，每个视野拍摄405nm和488nm激发波长下的图像；DAPI染色后细胞核在405nm激发波长下呈蓝色，细胞中表达的GFP荧光蛋白在488nm激发波长下呈绿色；系统通过识别蓝色区域精确定位细胞核位置，以细胞核外扩固定值识别细胞范围，并计算每个细胞范围内绿色荧光值，最后计算25个视野中所有标记细胞范围内的绿色荧光强度的平均值。
本实施例的测试思路如图5所示。启动子发生甲基化的基因无法发生转录和蛋白表达，因此当pEGFP-N1荧光报告质粒发生100％甲基化时，观察不到GFP绿色荧光蛋白在细胞中的表达；而当pEGFP-N1荧光报告质粒发生部分甲基化时，GFP绿色荧光蛋白在细胞中低表达；而如果Mini-CRISD可以对启动子区域去甲基化，则GFP绿色荧光蛋白能够重新表达。因此， 在本实施例中，GFP荧光强度与质粒甲基化程度呈反向线性相关。
为了对Mini-CRISD的去甲基化效果进行定量，我们以50％甲基化的荧光报告质粒为例。如图6所示，50％甲基化的荧光报告质粒在细胞中的荧光强度很低；加入Mini-CRISD后，Mini-CRISD在靶向CMV启动子区域的sgRNA引导下对该区域特性的去甲基化，显著提高了GFP荧光蛋白的表达和荧光强度。而在不加入sgRNA的对照组，即使有Mini-CRISD融合蛋白也没有提高荧光强度，证实观测到的GFP表达量增加(即荧光强度增强)是因为靶向的DNA去甲基化所致。
实施例5：
在293T细胞中使用Mini-CRISD方法对甲基化的荧光报告质粒进行去甲基化测试。详细步骤如下。
1)293T细胞以12000个/孔的密度均匀接种于96孔板，使用DMEM高糖培养基(含10％胎牛血清)，在37℃、5％CO 2的恒温培养箱中培养。
2)24小时后使用lipofectamine 3000向293T细胞同时转染从实施例2中获得的75％甲基化的荧光报告质粒与实施例3中获得的Mini-CRISD表达质粒各50ng(携带P CMV-sgRNA3、4、5、6中的一个)。对照组为同时转染50％甲基化的荧光报告质粒和Mini-CRISD-dead(去甲基化失活突变)质粒各50ng。
3)48小时后去除培养基，PBS漂洗2遍后用4％多聚甲醛固定，15分钟后吸去4％多聚甲醛，PBS漂洗一遍，每孔加入30μL DAPI染色液，5分钟后吸去染色液，PBS漂洗2遍，每孔加入100μL PBS。
4)使用Thermo Scientific Cell insight CX7-LZR高内涵分析系统采集图像。96孔板每孔选取25个视野，每个视野拍摄405nm和488nm激发波长下的图像；DAPI染色后细胞核在405nm激发波长下呈蓝色，细胞中表达的GFP荧光蛋白在488nm激发波长下呈绿色；系统通过识别蓝色区域精确定位细胞核位置，以细胞核外扩固定值识别细胞范围。然后取25个视野中的5个随机视野，对三个复孔进行相同的方式选取，使用ImageJ软件计算平均荧光强度，即：平均荧光强度(Mean)＝该区域荧光强度总和(IntDen)/该区域面积(Area)。
为了对Mini-CRISD的去甲基化效果进行定量，我们以75％甲基化的荧光报告质粒为例。结果如图7所示，75％甲基化的荧光报告质粒在细胞中的荧光强度较低；加入Mini-CRISD后，Mini-CRISD在靶向CMV启动子区域的sgRNA引导下对该区域特性的去甲基化，显著提高了GFP荧光蛋白的表达和荧光强度。而Mini-CRISD-dead因为失活了去甲基化的活性位点，即使加入了靶向启动子的sgRNA也没有提高荧光强度，进一步证实观测到的GFP表达量的增加(即 荧光强度的增强)是因为靶向的DNA去甲基化所致。
实施例6
通过Mini-CRISD方法靶向抑癌基因RASSF1A启动子区域进行去甲基化，从而提高肺癌细胞中RASSF1A的表达水平。包括以下步骤。
1)构建靶向人因组中抑癌基因RASSF1A启动子的Mini-CRISD质粒。选取RASSF1A启动子上符合CjCas9PAM序列要求的识别序列6条(见下表)，DNA靶点在基因组和RASSF1A启动子上的相对位置如图8所示。其中P RASSF1A-sgRNA-8使用的是5’-NNNNACA-3’PAM位点，其余sgRNA使用的是5’-NNNVRYAC-3’PAM位点。
按照和实施例3中相同的构建方法，用上述识别序列替换Mini-CRISD载体质粒上的空白序列，获得6个靶向人抑癌基因RASSF1A启动子的Mini-CRISD表达质粒(携带靶向人抑癌基因RASSF1A启动子的sgRNA)。
P RASSF1A-sgRNA-3 TTCCTTCCCTCCTTCGTCCCCT(SEQ ID NO.15)
P RASSF1A-sgRNA-4 GCTTGCTAGCGCCCAAAGCCAG(SEQ ID NO.16)
P RASSF1A-sgRNA-5 CTGAGCTCATTGAGCTGCGGGA(SEQ ID NO.17)
P RASSF1A-sgRNA-6 CCCCAGATGAAGTCGCCACAGA(SEQ ID NO.18)
P RASSF1A-sgRNA-7 TGCGACAAGGGATAAACCATTT(SEQ ID NO.19)
P RASSF1A-sgRNA-8 CCAGGGACCAGCTGCCGTGTGG(SEQ ID NO.20)
2.人肺癌细胞A549和H1299的培养
A549细胞以25000个/孔的密度均匀接种于6孔板，使用Ham's F-12K培养基(含10％FBS)；H1299细胞以30000个/孔的密度均匀接种于6孔板，使用RPMI-1640培养基(含10％FBS)。在37℃、5％CO 2环境的恒温培养箱中培养。
3.人肺癌细胞中RASSF1A启动子的去甲基化和RASSF1A表达的检测。
24小时后更换为新鲜培养基并进行转染实验。使用lipofectamine 3000向A549或H1299细胞转染1ug的Mini-CRISD表达质粒(携带P RASSF1A-sgRNA3、4、5、6、7、8中的一个)。6小时后更换为新的培养基。对照组使用Mini-CRISD-dead(去甲基化失活突变)质粒1ug。
其中A549细胞实验中还使用了DNA甲基化小分子抑制剂地西他滨作为一组阳性对照，将地西他滨(Sigma，货号A3656)溶于DMSO制成10mM母液，每2mL细胞培养基加入2ul地西他滨母液使得地西他滨终浓度10uM、每24小时更换新的含地西他滨的培养基。
48-72小时后使用SteadyPure通用型RNA提取试剂盒(广州瑞真，货号AG21022)提取转染细胞的总RNA，并用Evo M-MLV反转录试剂盒(广州瑞真，货号AG11711)和SYBR Green  Pro Taq HS预混型qPCR试剂盒(广州瑞真，货号AG11702)进行RT-qPCR实验，检测RASSF1A基因的表达。qPCR检测RASSF1A的引物为：GAAGTCATTGAGGCCCTGCT和ATCATCCAACAGCTTCCGCA。
结果如图9所示，Mini-CRISD在A549和H1299细胞中靶向抑癌基因RASSF1A启动子区域并去甲基化，使肺癌细胞中的RASSF1A基因得到了明显的提升，促进了RASSF1A基因的表达。Mini-CRISD靶向去甲基化的效果(基于RASSF1A基因表达水平的提升)明显优于非靶向的小分子抑制剂(地西他滨)。
实施例7：通过Mini-CRISD方法靶向提高抑癌基因RASSF1A表达水平，促进肺癌细胞的死亡。
1.按实施例6中相同的方法向A549肺癌细胞中转染1ug的Mini-CRISD表达质粒(携带P RASSF1A-sgRNA7)。转染48-72小时后，对A549细胞进行Calcein-AM/PI活细胞/死细胞双染色检测。
2.从低温冰箱内取出10×Assay Buffer，用去离子水做10倍稀释以得到1×Assay Buffer。取5μL Calcein-AM溶液(2mM)和15μL PI溶液(1.5mM)加入5mL 1×Assay Buffer，充分混匀获得染色液。
3.去除培养基，用PBS清洗细胞2次，用1×Assay Buffer清洗细胞2次。取500μL染色工作液加入贴壁细胞的共聚焦皿中心，避光37℃孵育15min。
4.激光共聚焦显微镜下使用488nm激发光检测活细胞(绿色荧光)、535nm激发光检测死细胞(红色荧光)。Calcein-AM本身不发荧光，进入细胞后被细胞内酯酶剪切形成膜非渗透性的Calcein滞留在细胞内并发出强绿色荧光；而死细胞缺乏酯酶活性，因此Calcein-AM仅标记活细胞。碘化丙啶(Propidium iodide，PI)不能穿过活细胞的细胞膜，仅能穿过死细胞膜到达细胞核，并嵌入细胞DNA双螺旋从而产生红色荧光，因此PI仅标记死细胞。
结果如图10所示，Mini-CRISD在A549细胞中靶向抑癌基因RASSF1A启动子区域并去甲基化，促进了RASSF1A基因的表达，从而导致A549细胞产生了死亡，相比对照组Mini-CRISD-dead组和未转染组具有显著性差别。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (17)

  1. 一种融合蛋白，其特征在于，其包括截短后的CjCas9，截短后的ROS1，具有连接子的中间序列，所述中间序列将截短后的CjCas9、截短后的ROS1连接，其中,所述截短后的CjCas9为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用连接子连接、并进行D8A点突变的氨基酸片段；所述截短后的ROS1为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用连接子连接的氨基酸片段，且所述ROS1蛋白的971位的氨基酸为天冬氨酸。
  2. 根据权利要求1所述的融合蛋白，其特征在于，所述截短后的CjCas9的氨基酸组成从N端到C端依次为：
    SEQ ID NO.1或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、连接子、和SEQ ID NO.2或其经过一个或多个氨基酸突变、取代但生物活性不变的序列。
  3. 根据权利要求1或2所述的融合蛋白，其特征在于，所述截短后的ROS1的氨基酸组成从N端到C端依次为:
    SEQ ID NO.3或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、连接子、和SEQ ID NO.4或其经过一个或多个氨基酸突变、取代但生物活性不变的序列。
  4. 根据权利要求1-3任一项所述的融合蛋白，其特征在于，所述融合蛋白从N端到C端的包括：
    SEQ ID NO.1或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、连接子、和SEQ ID NO.2或其经过一个或多个氨基酸突变、取代但生物活性不变的序列；
    中间序列；
    SEQ ID NO.3或其经过一个或多个氨基酸突变、取代但生物活性不变的序列、连接子、和SEQ ID NO.4或其经过一个或多个氨基酸突变、取代但生物活性不变的序列。
  5. 根据权利要求1所述的融合蛋白，其特征在于，所述中间序列包括NLS核定位信号肽、酶切位点和连接子。
  6. 根据权利要求1所述的融合蛋白，其特征在于，所述融合蛋白的截短后的CjCas9的前端还具有前序序列，所述前序序列包括NLS核定位信号肽和/或酶切位点。
  7. 编码权利要求1-6任一项融合蛋白的核苷酸序列，或为上述核苷酸序列且具有一个或多个核苷酸突变，但编码相同氨基酸的核苷酸序列。
  8. 表达权利要求1-6任一项所述融合蛋白的表达载体。
  9. 根据权利要求8所述的表达载体，其特征在于，所述载体还携带用于表达sgRNA的DNA核 苷酸序列，每个sgRNA序列包括骨架序列和识别靶DNA的识别序列。
  10. 一种靶向DNA去甲基化的试剂盒，其特征在于，其包括权利要求1-6任一项所述融合蛋白，或包括权利要求8或9所述表达载体。
  11. 根据权利要求10所述的DNA去甲基化的试剂盒，其特征在于，还包括sgRNA序列，每个sgRNA序列包括能与所述截短后的CjCas9结合的骨架序列和识别靶DNA的识别序列。
  12. 根据权利要求10所述的DNA去甲基化的试剂盒，其特征在于，所述sgRNA序列在权利要求9所述表达载体中表达获得；或所述sgRNA序列通过非权利要求9所述表达载体获得。
  13. 一种靶向DNA去甲基化方法，其特征在于，包括以下步骤：
    s1.构建表达载体；
    s2.获得sgRNA，所述sgRNA序列包括骨架序列和识别靶DNA的识别序列；
    s3.将所述表达载体和所述sgRNA导入含有靶DNA序列的目标物，进行靶向去甲基化。
  14. 权利要求1-6任一项融合蛋白或者权利要求8或9所述的表达载体在体内和/或离体和/或体外诱导的DNA去甲基化的应用。
  15. 权利要求1-6任一项融合蛋白或者权利要求8或9所述的表达载体在制备基于DNA去甲基化进行癌症治疗的药物中的应用。
  16. 权利要求1-6任一项融合蛋白或者权利要求8或9所述的表达载体在制备存在DNA异常甲基化现象的疾病治疗的药物中的应用。
  17. 一种融合对照蛋白，其特征在于，其包括、截短后的CjCas9、截短后的ROS1、具有连接子的中间序列，所述中间序列将截短后的CjCas9、截短后的ROS1连接，其中,所述截短后的CjCas9为:CjCas9蛋白被移除其481-640位氨基酸、剩余氨基酸用连接子连接、并进行D8A点突变的氨基酸片段；所述截短后的ROS1为:ROS1蛋白被移除其1-509位氨基酸和628-855位氨基酸、剩余氨基酸用连接子连接的氨基酸片段，且ROS1蛋白的971位的氨基酸不是天冬氨酸。
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