WO2016086768A1 - Chimeric nuclease for specifically recognizing and repairing β thalassemia beta-globin gene - Google Patents

Chimeric nuclease for specifically recognizing and repairing β thalassemia beta-globin gene Download PDF

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WO2016086768A1
WO2016086768A1 PCT/CN2015/094901 CN2015094901W WO2016086768A1 WO 2016086768 A1 WO2016086768 A1 WO 2016086768A1 CN 2015094901 W CN2015094901 W CN 2015094901W WO 2016086768 A1 WO2016086768 A1 WO 2016086768A1
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protein
gene
nucleic acid
acid molecule
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纪家葵
林建飞
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清华大学
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Definitions

  • the invention belongs to the field of genetic engineering technology, and particularly relates to a chimeric nuclease which specifically recognizes and repairs the beta-globin gene of beta thalassemia.
  • Thalassemia is a high-risk, widespread genetic disease in China and around the world.
  • Normal hematopoietic stem cells have the ability to differentiate into blood cells.
  • Thalassemia patients have abnormal red blood cells, are fragile and easy to die, and their oxygen carrying capacity is insufficient. Therefore, hematopoietic stem cell transplantation (HSCT) is currently curing severe ⁇ -mediterranean.
  • the most effective method of anemia includes bone marrow hematopoietic stem cell transplantation (BMT), peripheral blood stem cell transplantation (PB SCT), and cord blood stem cell transplantation (CBSCT).
  • BMT bone marrow hematopoietic stem cell transplantation
  • PB SCT peripheral blood stem cell transplantation
  • CBSCT cord blood stem cell transplantation
  • Beta-thalassemia is a type of anemia caused by mutations in the beta globin gene.
  • Normal hemoglobin consists of two parts of beta globin and two parts of alpha globin. If the expression level of ⁇ -globin is too small, it will result in agglomeration of ⁇ -globin and cause symptoms such as hemolysis.
  • Beta globin is expressed by the beta gene located on chromosome 11 of the chromosome. The beta gene contains multiple mutation hotspots, and the mutation hotspots are different in different regions.
  • I-SceI is a very specific homing endonuclease. It is a very specific and very safe endonuclease. It can combine DNA fragments of specific sequences on one hand and cleavage in recognized DNA sequences on the other hand. DNA double-strand breaks, the 18 sites of recognition and cleavage have very strong specificity. In many previous reports, I-SceI has been shown to be less toxic to the human genome. In published studies, the modification of homing endonucleases has been screened for specific recognition in bacteria or yeast. Of the 18 bases recognized by I-SceI, -7 bases to +11 bases are very important. The crystal structure shows that the specific recognition of these 18 bases and I-SceI proteins is formed by direct contact of many phosphates and bases and the regulation of water molecules between the two.
  • TALEN is currently the easiest to construct nucleases, and TALE, which specifically binds to any DNA, confers higher homologous recombination repair rates to TALEN.
  • TALE has the characteristic of specifically recognizing longer DNA sequences.
  • the current protein analysis and crystallization results show that the DNA binding domain of TALEs protein is highly conserved and consists of repeat fragments containing approximately 33-35 amino acids. In-depth research found that this section of ammonia The base acid is only two amino acids that are different, and these two amino acids are exactly the bases that specifically recognize the DNA. These two variable amino acids are referred to as repeatvariable di-residues (RVD).
  • RVD repeatvariable di-residues
  • HD specifically recognizes C bases
  • NI recognizes A bases
  • NN recognizes G or A bases
  • NG recognizes T bases.
  • nucleases include zinc finger protein (ZFN), TALEN and CRIPAR/Cas9 systems, and the like.
  • ZFN zinc finger protein
  • TALEN zinc finger protein
  • CRIPAR/Cas9 CRIPAR/Cas9 systems
  • ZFN zinc finger protein
  • TALEN technology has the characteristics of easy assembly, fast speed and strong identification.
  • FokI FokI
  • the CRISPR/Cas9 system has the characteristics of simple operation, but its binding to nucleic acids is very non-specific, which will hinder its safe application in clinical gene therapy.
  • CRISPR/CAS9 is not suitable for human genome targeting and modification of mutant genes (Wu et al., nature Biotechnology, 2014). , doi: 10.1038/nbt. 2889; Liang et al, Protein Cell, 2015, 6(5): 363-372).
  • the protein provided by the present invention is as follows 1) or 2):
  • the fusion protein shown is a protein obtained by replacing the TALE protein FokI with the modified I-SceI protein;
  • the amino acid sequence of the TALE protein is the sequence 4 in the sequence listing;
  • amino acid sequence of the engineered I-SceI protein is amino acid sequence 706-940 of the sequence 2 in the sequence listing;
  • amino acid sequence of the protein shown is the amino acid sequence 706-940 from the N' end of the sequence 2 in the sequence listing.
  • Nucleic acid molecules encoding the above proteins are also within the scope of the invention.
  • the above nucleic acid molecule is a DNA molecule according to any one of the following 1) to 4):
  • the coding region is a fusion protein represented by the DNA molecule (1) shown in SEQ ID NO: 1 in the sequence listing. Code area);
  • the coding region is the coding region of the protein represented by the DNA molecule (2) represented by nucleotides 2116-2823 of SEQ ID NO: 1 in the sequence listing);
  • the above stringent conditions may be to hybridize and wash the membrane at 65 ° C in a DNA or RNA hybridization experiment using a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.
  • An expression cassette, recombinant vector, recombinant strain, transgenic cell line or recombinant strain containing the above DNA molecule is also within the scope of protection of the present invention.
  • the above recombinant vector is a recombinant vector obtained by inserting the above nucleic acid molecule into an expression vector.
  • the expression vector used in the embodiment of the present invention is a PCS2 vector
  • the recombinant vector PCS2-TALE-I-SceI is a vector obtained by inserting the nucleotide represented by the sequence 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector.
  • This vector expresses the fusion protein TALE-I-SceI.
  • the recombinant strain is a recombinant strain obtained by introducing the recombinant vector into a target bacteria.
  • the above nucleic acid molecule an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain is also useful for improving gene efficiency of cleavage, improving gene efficiency of repairing target or reducing cytotoxicity
  • the scope of the invention is protected.
  • the target gene is cleaved so that the target gene after cleavage is self-repaired in the cell, and finally the normal gene sequence is produced.
  • the gene of interest is the beta thalassemia beta-globin gene.
  • the above protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain are prepared for repairing a patient with ⁇ -thalassemia in vitro
  • the use of mutant products of genes carried by blood stem cells is also within the scope of the invention.
  • the gene is a ⁇ -thalassemia beta-globin gene.
  • Another object of the present invention is to provide a kit for cleavage of a gene of interest and/or repair of a gene of interest.
  • the kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a third object of the present invention is to provide a kit for improving the efficiency of gene gene for cleavage, improving the efficiency of repairing a gene of interest, or reducing cytotoxicity.
  • the kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a fourth object of the present invention is to provide a therapeutic or adjunctive treatment of a beta-thalassemia product.
  • the active ingredient of the product provided by the present invention is the above-mentioned protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
  • a fifth object of the present invention is to provide a mutant product for repairing a gene carried by a hematopoietic stem cell of a patient with ⁇ -thalassemia in vitro, wherein the active ingredient is the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, Recombinant bacteria, transgenic cell lines or recombinant bacteria.
  • the gene is a ⁇ -thalassemia beta-globin gene
  • the above products are drugs or kits.
  • Figure 1 shows a green fluorescent protein repair reporter system expressed in human cells.
  • Figure 2 is a scheme for engineering I-SceI to specifically recognize the beta-globin DNA sequence.
  • Figure 3 shows the position and screening results of the I-SceI protein modification.
  • Figure 4 is a schematic representation of the chimeric protein.
  • Figure 5 shows the targeting efficiency of the modified I-SceI protein and TALE-ISceI in the beta-globin DNA sequence, respectively.
  • Figure 6 shows the gene repair efficiency of the modified I-SceI protein and TALE-ISceI.
  • Figure 7 is a comparison of the cytotoxicity of the engineered I-SceI protein and TALE-ISceI.
  • Figure 8 shows the expression of the modified I-SceI protein and Tale-ISVBII by Western blot.
  • Example 1 Preparation of a chimeric nuclease that specifically recognizes and repairs the beta-globin gene of beta thalassemia
  • the homing endonuclease I-SceI uses a specific fluorescent reporter system in human cells to screen for DNA sequences that specifically recognize the proximity of the beta thalassemia mutation site (Fig. 1). From the structure of the current I-SceI, it can be found that certain amino acids and DNA sequences in I-SceI are in direct contact, and the sequence of bases can be gradually changed, and then these directly contacted amino acids are screened, and the most specific new binding is found. The binding site for the amino acid (see Figure 2).
  • ATAAC in DNA sequences is an important part of protein cleavage. When these bases are changed, the cleavage efficiency will be significantly reduced. It can be screened by changing the amino acid of I-SceI. To the I-SceI engineered enzyme that recognizes the new sequence.
  • T at position -7 is directly in contact with N152 amino acid.
  • the strategy is to sequentially change the amino acid of I-SceI that is in direct contact with the four bases of DNA, and it is hoped that a new I-SceI amino acid capable of specifically recognizing the ⁇ -globin gene can be found.
  • I-SceI first recognition site TAG changed it to AAA of ⁇ -globin gene, then point mutation of N152 amino acid of I-SceI, changed to 19 other amino acids, and then used eGFP reporting system to screen the most Highly efficient HDR amino acids.
  • I-SceI Fig.
  • a I-SceI-recognized original sequence and post-engineered sequence recognized
  • b the three-dimensional structure map shows the need for altered nucleic acid sites and The position of interaction of the protein
  • cf which is the result of four rounds of screening
  • the altered nucleotide position of the engineered I-SceI coding gene is sequence 1 in the sequence listing from the 5' end of the 5' end, 45, 144, 456, 579, modified I -SceI amino acid sequence altered amino acid position as sequence Sequence 2 in the table is from position 15, 48, 152, 193 at the N' end.
  • the GFP Gene Targeting Systems (see Figure 4). This system consists of three parts: the first part, the cell line is stably integrated with one unit of GFP gene by viral transfection, and the coding region of this gene is inserted into a target sequence of 18 bp which is gradually transformed into ⁇ -globin, so that GFP cannot be expressed normally. In the second part, the engineered endonuclease I-SceI was expressed by viral transfection in cells. In the third part, the donor is transfected into a donor sequence containing a truncated GFP (tGFP) sequence.
  • tGFP truncated GFP
  • the homing endonuclease When the homing endonuclease cleaves to form a DNA double-strand break, it can induce homologous recombination of the donor fragment and the endonuclease recognition fragment, repair the gene, and repair the eGFP gene into the correct sequence to express the eGFP protein. .
  • the relative content of eGFP protein-positive cells was detected by flow cytometry, etc., so the relative repair efficiency of I-SceI protein can be judged to find the most suitable I-SceI engineering enzyme. In the absence of I-SceI, homologous repair is very inefficient and it is generally believed that one of the 10 million cells will be repaired. In the presence of I-SceI, this efficiency can be significantly improved, reaching more than 1000 times, which is why the high specificity of the target protein is very important.
  • TALE Three pairs of homologous enzymes are currently found on the repeats of TALE: BsaI (multiple sites are recognizable), SpeI and NheI, and BspEI and XmalI found.
  • BsaI multiple sites are recognizable
  • SpeI and NheI the fragment can be ligated by T4 ligase, and the fragment can be identified.
  • Two bases Two bases.
  • a TALE plasmid sequence capable of recognizing the 15 bp globin gene sequence corresponding to the TALE amino acid was constructed.
  • the 53 bp globin sequence to be recognized was inserted into the coding region of the eGFP gene using the "green fluorescent protein gene targeting system", and the biological activity of the constructed TALE plasmid was verified.
  • the forward primer "CACCATGGCTCCAAAGAAGAAGCGTAAGGTA” and the reverse primer “GGATCCGGCAACGCGA TGGGATGTGC” were used to obtain a 2119 bp TALE amplification product;
  • the above TALE amplification product was digested with NcoI and BamHI, and the obtained digested product was ligated with the same digested PCS2 vector (addgene, plasmid #17095) to obtain an intermediate vector PCS2-TALE (for sequence 1 from 5'
  • PCS2-TALE for sequence 1 from 5'
  • the 1-2115 nucleotide of the terminal is inserted between the NcoI and BamHI restriction sites of the PCS2 vector, and the PCS2 vector contains a CMV promoter, which is capable of initiating expression of the gene of interest in various human cell lines;
  • the above I-SceI amplification product was digested with BamHI and XbaI, and the digested product was ligated with the same digested intermediate vector PCS2-TALE to obtain a recombinant vector;
  • the recombinant vector is a vector obtained by inserting the nucleotide shown in SEQ ID NO: 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector, and named PCS2-TALE-I-SceI, which expresses the fusion protein TALE.
  • PCS2-TALE-I-SceI which expresses the fusion protein TALE.
  • -I-SceI which is a chimeric nuclease, the amino acid sequence of which is sequence 2 in the sequence listing.
  • the nucleotide sequence shown in SEQ ID NO: 1 includes the engineered I-SceI encoding gene and the TALE encoding gene ligated upstream thereof.
  • the nucleotide sequence of the I-SceI encoding gene after the transformation is sequence 1 from the 5' end.
  • the nucleotide sequence of the TALE-encoding gene is sequence 1 from nucleotides 1-2115 of the 5' end.
  • Sequence 2 in the sequence listing is from the N-terminal 1-705 amino acid residue to TALE, and from the N-terminal amino acid residues 706-940 is I-SceI.
  • a recombinant vector expressing I-SceI was constructed, and the transformed I-SceI coding gene (sequence 1 from the 5' end 2116-2823 nucleotide) was inserted into the NcoI and XbaI restriction sites of the PCS2 vector, and was named as PCS2-I-SceI;
  • TALE-encoding recombinant vector was constructed, and the TALE-encoding gene (SEQ ID NO: 1 to 1-5115 from the 5' end) was inserted into the vector of the NcoI and XbaI cleavage sites of the PCS2 vector, Named PCS2-TALE.
  • the engineered fusion protein has the effect of cutting genomic DNA. Without providing a donor, DSB will be repaired by NEHJ repair in vivo, producing indel or mutations at the cleavage site.
  • the expression of the protein was identified by anti-flag antibody by immunofluorescence technique, and the flag was tagged on the PCS2 vector, so it can be used to detect the expression of the target protein.
  • the target proteins of the three transgenic cells were expressed. (See Figure 8, western blot.)
  • PCR amplification was performed with the forward primer "GCCTAGTACATTACTATTTG” and the reverse primer "ATTAGGCAGAATCCAGATGC” to obtain a 768 bp target fragment, which is a beta-globin gene partial fragment of beta thalassemia (beta-globin gene)
  • the length is sequence 6, and the nucleotide sequence of this partial fragment is sequence 6 from the 5' end of nucleotides 892-1565).
  • the PCR product was ligated to the T3 (full-scale gold product) vector, and the monoclonal was selected for Sanger sequencing.
  • the fusion protein TALE-I-SceI is much more efficient at cutting the target fragment than the I-SceI alone.
  • the PCS2-eGFP vector (purchased by Addgene) was used as a template, and PCR amplification was carried out using the forward primer "CACCATGGTGAGCAAGGGCGAGGAGC” and the reverse primer "CTACTTGTACAGCTCGTCCATGC” to obtain a 724 bp green fluorescent protein eGFP gene (SEQ ID NO: 7).
  • a forward primer "ACGTCCAGGAGCGCACCATCTAAAGGATAACAGGGTAATTCTTCAAGGACGACGGCA” and a reverse primer “CTACTTGTACAGCTCGTCCATGC” were used to obtain a 461 bp PCR product 2;
  • CACCATGGTGAGCAAGGGCGAGGAGC and CTACTTGTACAGCTCGTCCATGC were used as primers to obtain a 742 bp DNA fragment eGFP-1.
  • nucleotide sequence of the DNA fragment is SEQ ID NO: 3 in the sequence listing, wherein the nucleotides 299-316 from the 5' end are I-SceI protein recognition sequences.
  • the DNA fragment eGFP-1 prepared in the above 2) was inserted into a D-TOPO vector (topological enzyme recombination, purchased by Life Technology) to obtain a recombinant vector D-TOPO-eGFP-1;
  • the above recombinant vector D-TOPO-eGFP-1 was subjected to LR recombination, and the eGFP-1 sequence was recombined into a p2k viral vector (purchased from Life Technology) to obtain a reporter vector;
  • 293 cells were transfected with the reporter vector to obtain transgenic cells containing eGFP-1 as a reporter system.
  • PCS2-TALE-I-SceI plasmid prepared in Example 1 and the plasmid containing the donor fragment were co-transfected (mass ratio of plasmid: 1:1) transgenic cells containing eGFP-1; after transfection for 36 hours, cells were collected. , using flow cytometry analysis.
  • the plasmid containing the donor fragment is a vector obtained by inserting a donor fragment (nucleotide sequence is sequence 8) into a T3 (full-scale product) carrier.
  • the plasmid containing the donor fragment was separately transfected into control 2;
  • the PCS2-TALEN vector is a vector obtained by inserting the TALEN encoding gene shown in SEQ ID NO: 5 into the NcoI and XbaI cleavage sites of the PCS2 vector.
  • the transfection rate of transfected cells (TALE-ISVBII) transfected with PCS2-TALE-I-SceI vector was 0.21%.
  • the GFP positive cell rate in transgenic cells (ISVBII) infected with PCS2-I-SceI vector was 0.10%; the GFP positive cell rate in transgenic cells (TALEN) transfected with PCS2-TALEN vector was 0.22%; 0, it can be seen that TALE-ISVBII expression of TALE-ISceI protein can significantly improve the repair efficiency of 0.21%, ISVBII expression of I-SceI protein repair efficiency of 0.1%.
  • the fusion protein TALE-I-SceI has low cytotoxicity
  • Both ⁇ -H2AX and 53BP1 proteins are used to detect the biomarkers that produce DSB in cells, and are characterization proteins for cytotoxicity by detecting these two biomarker experiments. Immunofluorescence staining of ⁇ -H2AX and 53BP1 showed that the gene toxicity of TALEN was high.
  • the experimental method is as follows:
  • the cells were cultured for 36 hours.
  • the cells of the 6-well plate were subjected to immunofluorescence staining.
  • the experimental procedure can be briefly as follows: 4% formaldehyde for 15 min, 0.1% Triton X-100 for 10 min, 10% FBS for 1 h, anti- ⁇ -H2AX and 53BP1 protein antibodies were incubated for 1 h, washed with PBS for 3 min for 5 min, secondary antibody for 1 h, washed with PBS for 5 min, DAPI stained for 15 min, washed with PBS for 3 min for 5 min, and the resulting cells were used for microscopic observation or flow analysis.
  • A is the result of ⁇ -H2AX and b is the result of 53BP1. It can be seen that the transfected cells transfected with PCS2-TALE-I-SceI can be compared with the transfected cells transfected with PCS2-I-SceI. Significantly reduced cytotoxicity, reflected in a significant decrease in the expression of ⁇ -H2AX and 53BP1 protein levels.
  • the experiments of the present invention prove that the present invention utilizes the low genotoxicity of homing endonuclease I-SceI and the high-efficiency targeting advantage of TALEN nuclease to construct a fusion endonuclease TALE-ISceI, which is a hematopoietic stem cell of a patient with ⁇ -thalassemia disease. Model to repair its mutant genes.
  • the enzyme plays a role in hematopoietic stem cells of patients with ⁇ -thalassemia in vitro and is expected to be widely used in clinical practice. At the same time, the enzyme can be screened so that the enzyme recognizes and repairs more nucleic acid fragments and promotes them to other genetic diseases.
  • the novel gene targeting technology is used to repair mutations in genes carried by hematopoietic stem cells of patients with ⁇ -thalassemia in vitro, and to differentiate the repaired hematopoietic stem cells into red blood cells capable of normally expressing ⁇ -globin.
  • Direct gene repair on hematopoietic stem cells has the following advantages: avoidance of iPS transgene instability and possible carcinogenicity of iPS cells, while avoiding immunological rejection of transplanted cells. Therefore, the acquisition of this engineered enzyme provides a practical basis for the radical cure of patients with beta thalassemia.

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Abstract

Disclosed is chimeric nuclease TALE-ISceI, in which FokI in TALEN is replaced with fusion protein obtained by transformed I-SceI. The chimeric nuclease TALE-ISceI can specifically recognize and repair a β thalassemia β-globin gene and can be used for treating β-thalassemia.

Description

特异识别并修复β地中海贫血症beta-globin基因的嵌合核酸酶A chimeric nuclease that specifically recognizes and repairs the beta-globin gene of beta thalassemia 技术领域Technical field
本发明属于基因工程技术领域，具体涉及特异识别并修复β地中海贫血症beta-globin基因的嵌合核酸酶。The invention belongs to the field of genetic engineering technology, and particularly relates to a chimeric nuclease which specifically recognizes and repairs the beta-globin gene of beta thalassemia.
背景技术Background technique
地中海贫血症是中国及全球高发、分布广泛的遗传疾病。正常的造血干细胞具备分化为血液细胞能力，地中海贫血患者，其红细胞异常、脆弱且容易死亡，其带氧能力亦不足，因此造血干细胞移植(Hematopoietic stem cell transplantation,HSCT)是目前根治重型β-地中海贫血最有效的方法，它包括骨髓造血干细胞移植(BMT)、外周血干细胞移植(PB SCT)和脐血干细胞移植(CBSCT)。但是，供体缺乏和排异反应严重阻滞了此方法的广泛应用。Thalassemia is a high-risk, widespread genetic disease in China and around the world. Normal hematopoietic stem cells have the ability to differentiate into blood cells. Thalassemia patients have abnormal red blood cells, are fragile and easy to die, and their oxygen carrying capacity is insufficient. Therefore, hematopoietic stem cell transplantation (HSCT) is currently curing severe β-mediterranean. The most effective method of anemia includes bone marrow hematopoietic stem cell transplantation (BMT), peripheral blood stem cell transplantation (PB SCT), and cord blood stem cell transplantation (CBSCT). However, donor deficiency and rejection have severely retarded the widespread use of this method.
β-地中海贫血是一类由于β珠蛋白基因突变引起的贫血。正常的血红蛋白由2份β珠蛋白和2份α珠蛋白组成四聚体。如果β珠蛋白的表达量过少，将导致α珠蛋白的聚体并导致溶血等症状。β珠蛋白由位于第11对染色体的β基因表达。β基因含有多个突变热点，不同的地区，突变热点有所不同。Beta-thalassemia is a type of anemia caused by mutations in the beta globin gene. Normal hemoglobin consists of two parts of beta globin and two parts of alpha globin. If the expression level of β-globin is too small, it will result in agglomeration of α-globin and cause symptoms such as hemolysis. Beta globin is expressed by the beta gene located on chromosome 11 of the chromosome. The beta gene contains multiple mutation hotspots, and the mutation hotspots are different in different regions.
I-SceI是非常特殊的归巢内切酶，是一个特异性非常高并且十分安全的内切酶，一方面可以结合特异序列的DNA片段，另一方面能在识别的DNA序列中进行切割产生DNA双链断裂，其识别和切割的18个位点具有非常强的特异性。在前期许多报道中，已显示I-SceI对人类基因组的低毒性，已发表的研究中，对归巢核酸内切酶的改造多在细菌或酵母中筛选其特导性识別。I-SceI的识别的18个碱基中,﹣7位碱基到+11位碱基都非常重要。结晶结构显示，这18个碱基和I-SceI蛋白的特异性识别通过许多磷酸和碱基的直接接触以及水分子在两者之间的调节作用形成的。I-SceI is a very specific homing endonuclease. It is a very specific and very safe endonuclease. It can combine DNA fragments of specific sequences on one hand and cleavage in recognized DNA sequences on the other hand. DNA double-strand breaks, the 18 sites of recognition and cleavage have very strong specificity. In many previous reports, I-SceI has been shown to be less toxic to the human genome. In published studies, the modification of homing endonucleases has been screened for specific recognition in bacteria or yeast. Of the 18 bases recognized by I-SceI, -7 bases to +11 bases are very important. The crystal structure shows that the specific recognition of these 18 bases and I-SceI proteins is formed by direct contact of many phosphates and bases and the regulation of water molecules between the two.
TALEN是目前最容易构建核酸酶，其中可特异结合任意DNA的TALE赋予TALEN较高的同源重组修复率。TALE具有特异性识别较长DNA序列的特点，通过目前的蛋白分析和结晶结果发现，TALEs蛋白的DNA结合域是高度保守的由大约含有33-35个氨基酸的重复片段组成。深入的研究发现，这一段氨 基酸只有两个氨基酸是不同的，而这两个氨基酸正好是特异性识别DNA的碱基。这两个可变的氨基酸被称为重复序列可变的双氨基酸残基(repeatvariable di-residues，RVD)。这种对应关系一共有五种，分别是：HD(分别为氨基酸)特异识别C碱基，NI识别A碱基，NN识别G或A碱基，NG识别T碱基，这种识别还包括某些非特异的配对，如NS可以识别A、T、G、C中的任一种。TALEN is currently the easiest to construct nucleases, and TALE, which specifically binds to any DNA, confers higher homologous recombination repair rates to TALEN. TALE has the characteristic of specifically recognizing longer DNA sequences. The current protein analysis and crystallization results show that the DNA binding domain of TALEs protein is highly conserved and consists of repeat fragments containing approximately 33-35 amino acids. In-depth research found that this section of ammonia The base acid is only two amino acids that are different, and these two amino acids are exactly the bases that specifically recognize the DNA. These two variable amino acids are referred to as repeatvariable di-residues (RVD). There are five such correspondences: HD (respectively amino acid) specifically recognizes C bases, NI recognizes A bases, NN recognizes G or A bases, and NG recognizes T bases. Some non-specific pairings, such as NS, can identify any of A, T, G, and C.
目前，常用的核酸酶包括锌指蛋白(ZFN)，TALEN和CRIPAR/Cas9***等。其中ZFN目前只能识别少数的位点，应用起来会有比较大的限制；TALEN技术具有组装容易，速度快，识别强的特点，但是由于TALEN中含有FokI，具有识别上的非特异性，造成一定的脱靶性；CRISPR/Cas9***具有操作简单的特点，但是，其和核酸的结合具有非常大的非特异性，这将阻碍其安全的应用于临床基因治疗。近期所发表的多篇论文，包括利用CRISPR/CAS9于人类胚胎基因打靶的研究结果，都充份显示CRISPR/CAS9还不适用于人类基因组打靶及修正突变基因(Wu et al.,nature Biotechnology,2014,doi:10.1038/nbt.2889；Liang et al,Protein Cell,2015,6(5):363-372)。Currently, commonly used nucleases include zinc finger protein (ZFN), TALEN and CRIPAR/Cas9 systems, and the like. Among them, ZFN can only identify a few sites, and there are relatively large restrictions on application. TALEN technology has the characteristics of easy assembly, fast speed and strong identification. However, because TALEN contains FokI, it has non-specificity in recognition, which causes certain The off-target property; the CRISPR/Cas9 system has the characteristics of simple operation, but its binding to nucleic acids is very non-specific, which will hinder its safe application in clinical gene therapy. A number of recent papers, including the use of CRISPR/CAS9 in human embryonic gene targeting, have fully shown that CRISPR/CAS9 is not suitable for human genome targeting and modification of mutant genes (Wu et al., nature Biotechnology, 2014). , doi: 10.1038/nbt. 2889; Liang et al, Protein Cell, 2015, 6(5): 363-372).
发明公开Invention disclosure
本发明的一个目的是提供了特异识别并β地中海贫血症beta-globin基因的嵌合核酸酶。It is an object of the present invention to provide a chimeric nuclease that specifically recognizes the beta thalassemia beta-globin gene.
本发明提供的蛋白质，为如下1)或2)：The protein provided by the present invention is as follows 1) or 2):
1)所示的融合蛋白，为将TALE蛋白FokI替换成改造后I-SceI蛋白得到的蛋白；1) The fusion protein shown is a protein obtained by replacing the TALE protein FokI with the modified I-SceI protein;
所述TALE蛋白的氨基酸序列为序列表中序列4；The amino acid sequence of the TALE protein is the sequence 4 in the sequence listing;
所述改造后I-SceI蛋白的氨基酸序列为序列表中序列2自N’末端第706-940位氨基酸；The amino acid sequence of the engineered I-SceI protein is amino acid sequence 706-940 of the sequence 2 in the sequence listing;
2)所示的蛋白的氨基酸序列为序列表中序列2自N’末端第706-940位氨基酸。2) The amino acid sequence of the protein shown is the amino acid sequence 706-940 from the N' end of the sequence 2 in the sequence listing.
1)所示的融合蛋白的氨基酸序列为序列表中序列2。1) The amino acid sequence of the fusion protein shown is Sequence 2 in the Sequence Listing.
编码上述的蛋白的核酸分子也是本发明保护的范围。Nucleic acid molecules encoding the above proteins are also within the scope of the invention.
上述核酸分子为如下1)-4)中任一所述的DNA分子：The above nucleic acid molecule is a DNA molecule according to any one of the following 1) to 4):
1)编码区为序列表中序列1所示的DNA分子(1)所示融合蛋白的编 码区)；1) The coding region is a fusion protein represented by the DNA molecule (1) shown in SEQ ID NO: 1 in the sequence listing. Code area);
2)编码区为序列表中序列1第2116-2823位核苷酸所示的DNA分子(2)所示蛋白的编码区)；2) the coding region is the coding region of the protein represented by the DNA molecule (2) represented by nucleotides 2116-2823 of SEQ ID NO: 1 in the sequence listing);
3)在严格条件下与1)或2)杂交且编码具有相同功能蛋白的DNA分子；3) hybridizing to 1) or 2) under stringent conditions and encoding a DNA molecule having the same functional protein;
4)与1)或2)具有90％以上同源性且编码具有相同功能蛋白的DNA分子。4) A DNA molecule having more than 90% homology with 1) or 2) and encoding a protein having the same function.
上述严格条件可为用0.1×SSPE(或0.1×SSC)，0.1％SDS的溶液，在DNA或者RNA杂交实验中65℃下杂交并洗膜。The above stringent conditions may be to hybridize and wash the membrane at 65 ° C in a DNA or RNA hybridization experiment using a solution of 0.1 x SSPE (or 0.1 x SSC), 0.1% SDS.
含有上述DNA分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌也是本发明保护的范围。An expression cassette, recombinant vector, recombinant strain, transgenic cell line or recombinant strain containing the above DNA molecule is also within the scope of protection of the present invention.
上述重组载体为将上述核酸分子***表达载体得到的重组载体。The above recombinant vector is a recombinant vector obtained by inserting the above nucleic acid molecule into an expression vector.
本发明的实施例采用的表达载体为PCS2载体，重组载体PCS2-TALE-I-SceI为将序列表中序列1所示的核苷酸***PCS2载体的NcoI和XbaI酶切位点得到的载体，该载体表达融合蛋白TALE-I-SceI。The expression vector used in the embodiment of the present invention is a PCS2 vector, and the recombinant vector PCS2-TALE-I-SceI is a vector obtained by inserting the nucleotide represented by the sequence 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector. This vector expresses the fusion protein TALE-I-SceI.
上述重组菌为将所述重组载体导入目的菌中得到的重组菌。The recombinant strain is a recombinant strain obtained by introducing the recombinant vector into a target bacteria.
上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在切割目的基因和/或修复目的基因中的应用也是本发明保护的范围；The use of the above protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain for cleavage of a gene of interest and/or a gene of interest is also within the scope of protection of the present invention;
或上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在提高切割目的基因效率、提高修复目的基因效率或降低细胞毒性中的应用也是本发明保护的范围。Or the above-mentioned protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain is also useful for improving gene efficiency of cleavage, improving gene efficiency of repairing target or reducing cytotoxicity The scope of the invention is protected.
切割目的基因是使切割后目的基因在细胞内进行自行修复，最后产生正常基因序列。The target gene is cleaved so that the target gene after cleavage is self-repaired in the cell, and finally the normal gene sequence is produced.
上述应用中，所述目的基因为β地中海贫血症beta-globin基因。In the above application, the gene of interest is the beta thalassemia beta-globin gene.
上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在制备治疗或辅助治疗β-地中海贫血病产品中的应用也是本发明保护的范围。The use of the above proteins, the above nucleic acid molecules, expression cassettes containing the above nucleic acid molecules, recombinant vectors, recombinant bacteria, transgenic cell lines or recombinant bacteria for the preparation of therapeutic or adjuvant treatment of β-thalassemia products is also within the scope of the present invention.
上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在制备体外修复β-地中海贫血患者的造 血干细胞所携带基因的突变产品中的应用也是本发明保护的范围。The above protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain are prepared for repairing a patient with β-thalassemia in vitro The use of mutant products of genes carried by blood stem cells is also within the scope of the invention.
上述应用中，所述基因为β地中海贫血症beta-globin基因。In the above application, the gene is a β-thalassemia beta-globin gene.
本发明另一个目的是提供一种切割目的基因和/或修复目的基因的试剂盒。Another object of the present invention is to provide a kit for cleavage of a gene of interest and/or repair of a gene of interest.
本发明提供的试剂盒，包括上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。The kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
本发明第三个目的是提供一种提高切割目的基因效率、提高修复目的基因效率或降低细胞毒性的试剂盒。A third object of the present invention is to provide a kit for improving the efficiency of gene gene for cleavage, improving the efficiency of repairing a gene of interest, or reducing cytotoxicity.
本发明提供的试剂盒，包括上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。The kit provided by the present invention comprises the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
本发明第四个目的是提供一种治疗或辅助治疗β-地中海贫血病产品。A fourth object of the present invention is to provide a therapeutic or adjunctive treatment of a beta-thalassemia product.
本发明提供的产品，其活性成分为上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。The active ingredient of the product provided by the present invention is the above-mentioned protein, the above nucleic acid molecule, an expression cassette containing the above nucleic acid molecule, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain.
本发明第五个目的是提供一种体外修复β-地中海贫血患者的造血干细胞所携带基因的突变产品，其活性成分为上述的蛋白、上述核酸分子、含有上述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。A fifth object of the present invention is to provide a mutant product for repairing a gene carried by a hematopoietic stem cell of a patient with β-thalassemia in vitro, wherein the active ingredient is the above protein, the above nucleic acid molecule, an expression cassette containing the nucleic acid molecule, a recombinant vector, Recombinant bacteria, transgenic cell lines or recombinant bacteria.
上述产品中，所述基因为β地中海贫血症beta-globin基因；In the above product, the gene is a β-thalassemia beta-globin gene;
上述产品为药物或试剂盒。The above products are drugs or kits.
附图说明DRAWINGS
图1为在人体细胞中表达的绿色荧光蛋白修复报告***。Figure 1 shows a green fluorescent protein repair reporter system expressed in human cells.
图2为改造I-SceI为特异性识别beta-珠蛋白DNA序列的流程。Figure 2 is a scheme for engineering I-SceI to specifically recognize the beta-globin DNA sequence.
图3为I-SceI蛋白改造对应的位置和筛选结果。Figure 3 shows the position and screening results of the I-SceI protein modification.
图4为嵌合蛋白示意图。Figure 4 is a schematic representation of the chimeric protein.
图5为改造后I-SceI蛋白和TALE-ISceI分别在beta-珠蛋白DNA序列的打靶效率。Figure 5 shows the targeting efficiency of the modified I-SceI protein and TALE-ISceI in the beta-globin DNA sequence, respectively.
图6为改造后I-SceI蛋白和TALE-ISceI的基因修复效率。Figure 6 shows the gene repair efficiency of the modified I-SceI protein and TALE-ISceI.
图7为改造后I-SceI蛋白和TALE-ISceI的细胞毒性比较。Figure 7 is a comparison of the cytotoxicity of the engineered I-SceI protein and TALE-ISceI.
图8为利用Western blot检测改造后I-SceI蛋白和Tale-ISVBII的表达。Figure 8 shows the expression of the modified I-SceI protein and Tale-ISVBII by Western blot.
实施发明的最佳方式The best way to implement the invention
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。The experimental methods used in the following examples are conventional methods unless otherwise specified.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
实施例1、特异性识别并修复β地中海贫血症beta-globin基因的嵌合核酸酶的制备Example 1. Preparation of a chimeric nuclease that specifically recognizes and repairs the beta-globin gene of beta thalassemia
一、归巢内切酶I-SceI改造I. Retrofit of homing endonuclease I-SceI
归巢内切酶I-SceI在人类细胞中，利用特殊的荧光报告***，来筛选特异识别β地中海贫血突变位点邻近的DNA序列(图1)。由目前的I-SceI的结构可以发现，I-SceI中的某些氨基酸和DNA序列是直接接触，可以通过逐渐改变碱基的序列，然后筛选这些直接接触的氨基酸，并找到结合最特异的新的氨基酸的结合位点(见图2)。The homing endonuclease I-SceI uses a specific fluorescent reporter system in human cells to screen for DNA sequences that specifically recognize the proximity of the beta thalassemia mutation site (Fig. 1). From the structure of the current I-SceI, it can be found that certain amino acids and DNA sequences in I-SceI are in direct contact, and the sequence of bases can be gradually changed, and then these directly contacted amino acids are screened, and the most specific new binding is found. The binding site for the amino acid (see Figure 2).
I-SceI和DNA序列的识别过程中，DNA序列中的ATAAC是蛋白切割的重要部分，改变这些碱基时，其切割效率将会发生显著的下降，可以通过改变I-SceI氨基酸的方法来筛选到能够识别新的序列的I-SceI工程酶。During the recognition of I-SceI and DNA sequences, ATAAC in DNA sequences is an important part of protein cleavage. When these bases are changed, the cleavage efficiency will be significantly reduced. It can be screened by changing the amino acid of I-SceI. To the I-SceI engineered enzyme that recognizes the new sequence.
在地中海贫血患者的β珠蛋白基因中，发现存在三段ATAAC序列，其中有一段ATAAC序列两边的序列和I-SceI识别的序列比较接近，同时也距离中国常见的β-地中海贫血症的突变位点之一，IVS-Ⅱ654(C→T)非常近，该突变位点在中国β-地中海贫血患者中约占24％，是第二大热点区域。因此，这段序列非常适合用来作为切割位点产生DNA双链断裂。通过对比发现，这段序列和I-SceI识别的序列有7个碱基的变化，其中4个碱基和I-SceI的氨基酸有直接的接触。如-7位的T直接和N152氨基酸接触。策略是依次改变与DNA的4个碱基直接接触的I-SceI的氨基酸，希望能找到能够特异性识别β珠蛋白基因的新的I-SceI氨基酸。I-SceI第一个识别位点TAG，把其改成β珠蛋白基因的AAA，然后对I-SceI的N152位氨基酸进行点突变，改变为其它19种氨基酸，再利用eGFP报告***筛选出最高效HDR氨基酸。依次类推，获得特异靶向β珠蛋白基因改造的I-SceI(图3，a，I-SceI识别的原始序列和改造后识别的序列；b,三维结构图展示的需要改变的核酸位点和蛋白的相互作用位置；c-f,分别是四轮筛选得到的结果)，改造的I-SceI编码基因的改变的核苷酸位置为序列表中的序列1自5’末端第45,144,456,579位，改造的I-SceI的氨基酸序列改变的氨基酸位置为序列 表中的序列2自N’末端第15,48,152,193位。In the beta globin gene of thalassemia patients, three ATAAC sequences were found, in which the sequence on both sides of an ATAAC sequence is close to the sequence recognized by I-SceI, and also from the common mutation of β-thalassemia in China. One of the points, IVS-II654 (C→T) is very close, and this mutation site accounts for about 24% of Chinese β-thalassemia patients, and is the second largest hot spot. Therefore, this sequence is very suitable for generating DNA double-strand breaks as a cleavage site. By comparison, this sequence and the sequence recognized by I-SceI have a 7 base change, of which 4 bases are in direct contact with the amino acid of I-SceI. For example, T at position -7 is directly in contact with N152 amino acid. The strategy is to sequentially change the amino acid of I-SceI that is in direct contact with the four bases of DNA, and it is hoped that a new I-SceI amino acid capable of specifically recognizing the β-globin gene can be found. I-SceI first recognition site TAG, changed it to AAA of β-globin gene, then point mutation of N152 amino acid of I-SceI, changed to 19 other amino acids, and then used eGFP reporting system to screen the most Highly efficient HDR amino acids. By analogy, I-SceI (Fig. 3, a, I-SceI-recognized original sequence and post-engineered sequence recognized) can be obtained by specifically targeting β-globin gene modification; b, the three-dimensional structure map shows the need for altered nucleic acid sites and The position of interaction of the protein; cf, which is the result of four rounds of screening), the altered nucleotide position of the engineered I-SceI coding gene is sequence 1 in the sequence listing from the 5' end of the 5' end, 45, 144, 456, 579, modified I -SceI amino acid sequence altered amino acid position as sequence Sequence 2 in the table is from position 15, 48, 152, 193 at the N' end.
对归巢蛋白酶改造结果的验证，利用“绿色荧光蛋白基因打靶***(GFP Gene Targeting Systems)”***进行(见图4)。这个***包括3个部分：第一部分，细胞系中通过病毒转染稳定整合一个单位的GFP基因，此基因编码区***了18bp逐步被改造为β-珠蛋白的靶序列，因而不能正常表达GFP。第二部分，细胞中通过病毒转染表达改造过的归巢内切酶I-SceI。第三部分，细胞中转染供体序列(donor sequence)，这一片断含有truncated GFP(tGFP)序列。在归巢内切酶产生切割形成DNA双链断裂时，能够诱导供体片段与内切酶识别片段进行同源重组，并对基因进行修复，eGFP基因被修复成正确的序列，从而表达eGFP蛋白。通过流式细胞技术等方法检测eGFP蛋白阳性细胞的相对含量，因此可以判断I-SceI蛋白的相对修复效率，从而找到最适合的I-SceI工程酶。在没有I-SceI的情况下，同源修复效率非常低，通常认为-1000万个细胞中会有一个细胞被修复。在有表达I-SceI的时候，这个效率能够显著的提高，达到1000倍以上，这也是为什么特异性高的打靶蛋白显得非常重要的原因。Verification of the results of homing protease engineering was performed using the "GFP Gene Targeting Systems" system (see Figure 4). This system consists of three parts: the first part, the cell line is stably integrated with one unit of GFP gene by viral transfection, and the coding region of this gene is inserted into a target sequence of 18 bp which is gradually transformed into β-globin, so that GFP cannot be expressed normally. In the second part, the engineered endonuclease I-SceI was expressed by viral transfection in cells. In the third part, the donor is transfected into a donor sequence containing a truncated GFP (tGFP) sequence. When the homing endonuclease cleaves to form a DNA double-strand break, it can induce homologous recombination of the donor fragment and the endonuclease recognition fragment, repair the gene, and repair the eGFP gene into the correct sequence to express the eGFP protein. . The relative content of eGFP protein-positive cells was detected by flow cytometry, etc., so the relative repair efficiency of I-SceI protein can be judged to find the most suitable I-SceI engineering enzyme. In the absence of I-SceI, homologous repair is very inefficient and it is generally believed that one of the 10 million cells will be repaired. In the presence of I-SceI, this efficiency can be significantly improved, reaching more than 1000 times, which is why the high specificity of the target protein is very important.
二、表达嵌合核酸酶TALE-I-SceI编码基因重组载体的制备2. Preparation of recombinant vector expressing chimeric nuclease TALE-I-SceI coding gene
将TALEN(氨基酸序列4及其编码基因的核苷酸序列5，购自addgene)，其由TALE和FokI组成)的FokI替换成I-SceI，构建同源重组修复率高、安全性高、脱靶效应低的融合型嵌合核酸酶TALE-ISceI(图4)。由于TALE的重复片段具有上述特性，可以对其进行特异性的改造使得其能识别β珠蛋白基因特定的DNA序列。目前TALE的改造方法都是利用同尾酶的方法，依据所需识别的DNA序列，将相对应的氨基酸片段连接起来。在TALE的重复片段上目前共发现三对同尾酶：BsaI(可识别多个位点)，SpeI和NheI以及发现的BspEI和XmalI。利用同尾酶的性质，可以按照想要的顺序，利用双酶切的方法，产生一个具有相同粘性末端的载体片段和DNA片段，再用T4连接酶将这个片段连接起来，此片段就可以识别两个碱基。利用这种方法，依据靶向β珠蛋白基因的序列，构建出可以识别15bp珠蛋白基因序列，对应TALE氨基酸的TALE质粒序列。利用“绿色荧光蛋白基因打靶***”，将所需要识别的53bp珠蛋白序列***到eGFP基因编码区，验证构建的TALE质粒其生物学活性。 Replacing FokI of TALEN (amino acid sequence 4 and its coding gene nucleotide sequence 5, purchased from addgene), which consists of TALE and FokI, with I-SceI, constructing homologous recombination with high repair rate, high safety, off-target The fusion-type chimeric nuclease TALE-ISceI with low effect (Fig. 4). Since the repeat fragment of TALE has the above characteristics, it can be specifically modified such that it can recognize the DNA sequence specific to the β-globin gene. At present, the TALE modification method utilizes the same tail enzyme method to link the corresponding amino acid fragments according to the DNA sequence to be recognized. Three pairs of homologous enzymes are currently found on the repeats of TALE: BsaI (multiple sites are recognizable), SpeI and NheI, and BspEI and XmalI found. By using the properties of the homologous enzyme, a vector fragment and a DNA fragment having the same cohesive end can be produced by a double digestion method in the desired order, and the fragment can be ligated by T4 ligase, and the fragment can be identified. Two bases. Using this method, based on the sequence targeting the β-globin gene, a TALE plasmid sequence capable of recognizing the 15 bp globin gene sequence corresponding to the TALE amino acid was constructed. The 53 bp globin sequence to be recognized was inserted into the coding region of the eGFP gene using the "green fluorescent protein gene targeting system", and the biological activity of the constructed TALE plasmid was verified.
表达嵌合核酸酶TALE-I-SceI编码基因重组载体PCS2-TALE-I-SceI的制备方法具体如下：The preparation method of the chimeric nuclease TALE-I-SceI encoding gene recombinant vector PCS2-TALE-I-SceI is as follows:
1、以TALE片段为模板，用正向引物“CACCATGGCTCCAAAGAAGAAGCGTAAGGTA”和反向引物“GGATCCGGCAACGCGA TGGGATGTGC”扩增，得到2119bp的TALE扩增产物；1. Using the TALE fragment as a template, the forward primer "CACCATGGCTCCAAAGAAGAAGCGTAAGGTA" and the reverse primer "GGATCCGGCAACGCGA TGGGATGTGC" were used to obtain a 2119 bp TALE amplification product;
2、用NcoI和BamHI酶切上述TALE扩增产物，得到的酶切产物与经过同样酶切的PCS2载体(addgene,plasmid#17095)连接，得到中间载体PCS2-TALE(为将序列1自5’末端第1-2115位核苷酸***PCS2载体的NcoI和BamHI酶切位点间得到)，PCS2载体含有CMV启动子，该启动子能够启动目的基因在多种人类细胞系内的表达；2. The above TALE amplification product was digested with NcoI and BamHI, and the obtained digested product was ligated with the same digested PCS2 vector (addgene, plasmid #17095) to obtain an intermediate vector PCS2-TALE (for sequence 1 from 5' The 1-2115 nucleotide of the terminal is inserted between the NcoI and BamHI restriction sites of the PCS2 vector, and the PCS2 vector contains a CMV promoter, which is capable of initiating expression of the gene of interest in various human cell lines;
3、以改造后的I-SceI为模板，用正向引物“GGATCCATGAAAAACATCAAAAAAAACC”和反向引物“TCTAGATTACTTAAGAAAAGTTTCGGAG”扩增，得到720bp的I-SceI扩增产物；3. Using the modified I-SceI as a template, the forward primer "GGATCCATGAAAAACATCAAAAAAAACC" and the reverse primer "TCTAGATTACTTAAGAAAAGTTTCGGAG" were amplified to obtain a 720 bp I-SceI amplification product;
用BamHI和XbaI酶切上述I-SceI扩增产物，得到酶切产物与经过同样酶切的中间载体PCS2-TALE连接，得到重组载体；The above I-SceI amplification product was digested with BamHI and XbaI, and the digested product was ligated with the same digested intermediate vector PCS2-TALE to obtain a recombinant vector;
经过测序，该重组载体为将序列表中序列1所示的核苷酸***PCS2载体的NcoI和XbaI酶切位点得到的载体，命名为PCS2-TALE-I-SceI，该载体表达融合蛋白TALE-I-SceI，即为嵌合核酸酶，该融合蛋白的氨基酸序列为序列表中序列2。After sequencing, the recombinant vector is a vector obtained by inserting the nucleotide shown in SEQ ID NO: 1 in the sequence table into the NcoI and XbaI cleavage sites of the PCS2 vector, and named PCS2-TALE-I-SceI, which expresses the fusion protein TALE. -I-SceI, which is a chimeric nuclease, the amino acid sequence of which is sequence 2 in the sequence listing.
其中序列表中序列1所示的核苷酸包括改造后I-SceI编码基因和连接在其上游的TALE编码基因，改造后I-SceI编码基因的核苷酸序列为序列1自5’末端第2116-2823位核苷酸，TALE编码基因的核苷酸序列为序列1自5’末端第1-2115位核苷酸。The nucleotide sequence shown in SEQ ID NO: 1 includes the engineered I-SceI encoding gene and the TALE encoding gene ligated upstream thereof. The nucleotide sequence of the I-SceI encoding gene after the transformation is sequence 1 from the 5' end. At nucleotides 2116-2823, the nucleotide sequence of the TALE-encoding gene is sequence 1 from nucleotides 1-2115 of the 5' end.
序列表中序列2自N末端第1-705位氨基酸残基为TALE，自N末端第706-940位氨基酸残基为I-SceI。 Sequence 2 in the sequence listing is from the N-terminal 1-705 amino acid residue to TALE, and from the N-terminal amino acid residues 706-940 is I-SceI.
构建表达I-SceI的重组载体，将改造后I-SceI编码基因(序列1自5’末端第2116-2823位核苷酸)***PCS2载体的NcoI和XbaI酶切位点得到的载体，命名为PCS2-I-SceI；A recombinant vector expressing I-SceI was constructed, and the transformed I-SceI coding gene (sequence 1 from the 5' end 2116-2823 nucleotide) was inserted into the NcoI and XbaI restriction sites of the PCS2 vector, and was named as PCS2-I-SceI;
构建表达TALE的重组载体，将TALE编码基因(序列1自5’末端第1-2115位核苷酸)***PCS2载体的NcoI和XbaI酶切位点得到的载体， 命名为PCS2-TALE。A TALE-encoding recombinant vector was constructed, and the TALE-encoding gene (SEQ ID NO: 1 to 1-5115 from the 5' end) was inserted into the vector of the NcoI and XbaI cleavage sites of the PCS2 vector, Named PCS2-TALE.
实施例2、嵌合核酸酶TALE-I-SceI的功能验证Example 2 Functional verification of chimeric nuclease TALE-I-SceI
一、提高人类基因组中基因打靶效率First, improve the efficiency of gene targeting in the human genome
经改造后的融合蛋白具有切割基因组DNA的作用，在不提供供体的情况下，DSB会通过体内的NEHJ的修复方式进行修复，在切割位点产生indel或者mutations。The engineered fusion protein has the effect of cutting genomic DNA. Without providing a donor, DSB will be repaired by NEHJ repair in vivo, producing indel or mutations at the cleavage site.
实验方法：experimental method:
1、将1×105个293ft细胞(life technology)铺在6孔板中，培养24h后，在细胞中分别转染1ug的由实施例1制备的PCS2-I-SceI、PCS2-TALE和PCS2-TALE-I-SceI质粒，得到转PCS2-I-SceI细胞、转PCS2-TALEN细胞和转PCS2-TALE-I-SceI细胞3种转基因细胞。1. 1×10 5 293 ft cells (life technology) were plated in 6-well plates, and after 24 hours of culture, 1 ug of PCS2-I-SceI, PCS2-TALE and PCS2 prepared in Example 1 were separately transfected into the cells. -TALE-I-SceI plasmid, three transgenic cells transfected into PCS2-I-SceI cells, transfected PCS2-TALEN cells and transfected PCS2-TALE-I-SceI cells.
转染24h后，利用免疫荧光技术，用anti-flag抗体鉴定蛋白的表达，在PCS2载体上带有flag标签，因此可以用来检测目的蛋白的表达，结果3种转基因细胞目的蛋白均表达。(见图8，western blot图。)After transfection for 24 hours, the expression of the protein was identified by anti-flag antibody by immunofluorescence technique, and the flag was tagged on the PCS2 vector, so it can be used to detect the expression of the target protein. As a result, the target proteins of the three transgenic cells were expressed. (See Figure 8, western blot.)
2、转染后培养36h，利用QIAGEN的基因组提取试剂盒提取基因组，步骤参见试剂盒说明书。2. After transfection, culture for 36 hours, and extract the genome using QIAGEN's genome extraction kit. For the procedure, refer to the kit instructions.
以基因组DNA为模板，用正向引物“GCCTAGTACATTACTATTTG”和反向引物“ATTAGGCAGAATCCAGATGC”进行PCR扩增，得到768bp目的片段，该目的片段为β地中海贫血症beta-globin基因部分片段(beta-globin基因全长为序列6，此部分片段核苷酸序列为序列6自5’末端第892-1565位核苷酸)。Using genomic DNA as a template, PCR amplification was performed with the forward primer "GCCTAGTACATTACTATTTG" and the reverse primer "ATTAGGCAGAATCCAGATGC" to obtain a 768 bp target fragment, which is a beta-globin gene partial fragment of beta thalassemia (beta-globin gene) The length is sequence 6, and the nucleotide sequence of this partial fragment is sequence 6 from the 5' end of nucleotides 892-1565).
3、将PCR的产物连接到T3(全式金公司产品)载体上，挑选单克隆，进行桑格测序。3. The PCR product was ligated to the T3 (full-scale gold product) vector, and the monoclonal was selected for Sanger sequencing.
切割目的片段的效率＝(含有突变或缺失目的片段的单克隆数/测序总克隆数)*100％Efficiency of cutting the fragment of interest = (number of clones containing mutation or deletion of the fragment of interest / total number of clones sequenced) * 100%
结果如图5所示，可以看出，转染PCS2-TALE-I-SceI后的细胞中切割目的片段的效率比转染PCS2-I-SceI显著提高，转染PCS2-I-SceI的细胞中切割目的片段的效率为10％，转染PCS2-TALE-I-SceI的细胞中切割目的片段的效率为13％；转染TALEN的细胞中切割目的片段的效率为15％。The results are shown in Fig. 5. It can be seen that the efficiency of cleavage of the desired fragment in cells transfected with PCS2-TALE-I-SceI was significantly higher than that of transfected PCS2-I-SceI, and transfected into cells of PCS2-I-SceI. The efficiency of cleavage of the fragment of interest was 10%, the efficiency of cleavage of the fragment of interest in cells transfected with PCS2-TALE-I-SceI was 13%, and the efficiency of cleavage of the fragment of interest in cells transfected with TALEN was 15%.
从上述可以看出，融合蛋白TALE-I-SceI对目的片段的切割效率远远高于单独的I-SceI。 As can be seen from the above, the fusion protein TALE-I-SceI is much more efficient at cutting the target fragment than the I-SceI alone.
二、提高人类基因组中基因同源重组修复效率Second, improve the efficiency of gene homologous recombination repair in the human genome
1、绿色荧光蛋白报告***的构建1. Construction of green fluorescent protein reporter system
1)、绿色荧光蛋白eGFP基因的获得1) Acquisition of green fluorescent protein eGFP gene
以PCS2-eGFP载体(Addgene购买)为模板，用正向引物“CACCATGGTGAGCAAGGGCGAGGAGC”和反向引物“CTACTTGTACAGCTCGTCCATGC”进行PCR扩增，得到724bp的绿色荧光蛋白eGFP基因(序列7)。The PCS2-eGFP vector (purchased by Addgene) was used as a template, and PCR amplification was carried out using the forward primer "CACCATGGTGAGCAAGGGCGAGGAGC" and the reverse primer "CTACTTGTACAGCTCGTCCATGC" to obtain a 724 bp green fluorescent protein eGFP gene (SEQ ID NO: 7).
2)、Overlap PCR的制备***I-SceI蛋白识别序列的eGFP片段eGFP-12) Preparation of Overlap PCR eGFP fragment eGFP-1 inserted into the I-SceI protein recognition sequence
以上述绿色荧光蛋白eGFP基因为模板，用正向引物“CACCATGGTGAGCAAGGGCGAGGAGC”和反向引物“TGCCGTCGTCCTTGAAGAATTACCCTGTTATCCTTTAGATGGTGCGCTCCTGGACGT”为引物，得到334p的PCR产物1；Using the green fluorescent protein eGFP gene as a template, using the forward primer "CACCATGGTGAGCAAGGGCGAGGAGC" and the reverse primer "TGCCGTCGTCCTTGAAGAATTACCCTGTTATCCTTTAGATGGTGCGCTCCTGGACGT" as primers, to obtain 334p PCR product 1;
以eGFP为模板，用正向引物“ACGTCCAGGAGCGCACCATCTAAAGGATAACAGGGTAATTCTTCAAGGACGACGGCA”和反向引物“CTACTTGTACAGCTCGTCCATGC”，得到461bp的PCR产物2；Using eGFP as a template, a forward primer "ACGTCCAGGAGCGCACCATCTAAAGGATAACAGGGTAATTCTTCAAGGACGACGGCA" and a reverse primer "CTACTTGTACAGCTCGTCCATGC" were used to obtain a 461 bp PCR product 2;
以PCR产物1和PCR产物2为模板，用CACCATGGTGAGCAAGGGCGAGGAGC和CTACTTGTACAGCTCGTCCATGC为引物，得到742bp的DNA片段eGFP-1。Using PCR product 1 and PCR product 2 as templates, CACCATGGTGAGCAAGGGCGAGGAGC and CTACTTGTACAGCTCGTCCATGC were used as primers to obtain a 742 bp DNA fragment eGFP-1.
经过测序，该DNA片段的核苷酸序列为序列表中序列3，其中自5’末端第299-316位核苷酸为I-SceI蛋白识别序列。After sequencing, the nucleotide sequence of the DNA fragment is SEQ ID NO: 3 in the sequence listing, wherein the nucleotides 299-316 from the 5' end are I-SceI protein recognition sequences.
3)、报告载体的制备3) Preparation of report carrier
将上述2)制备的DNA片段eGFP-1***D-TOPO载体(拓扑酶重组,Life Technology购买)中，得到重组载体D-TOPO-eGFP-1；The DNA fragment eGFP-1 prepared in the above 2) was inserted into a D-TOPO vector (topological enzyme recombination, purchased by Life Technology) to obtain a recombinant vector D-TOPO-eGFP-1;
将上述重组载体D-TOPO-eGFP-1经过LR重组，eGFP-1序列重组到p2k病毒载体(购自Life Technology)连接，得到报告载体；The above recombinant vector D-TOPO-eGFP-1 was subjected to LR recombination, and the eGFP-1 sequence was recombined into a p2k viral vector (purchased from Life Technology) to obtain a reporter vector;
利用报告载体转染293细胞，得到含有eGFP-1的转基因细胞，为报告***。293 cells were transfected with the reporter vector to obtain transgenic cells containing eGFP-1 as a reporter system.
2、将由实施例1制备的PCS2-TALE-I-SceI质粒和含有供体片段的质粒共同转染(质粒的质量比1:1)含有eGFP-1的转基因细胞；转染36h后，收集细胞，利用流式细胞仪分析。2. The PCS2-TALE-I-SceI plasmid prepared in Example 1 and the plasmid containing the donor fragment were co-transfected (mass ratio of plasmid: 1:1) transgenic cells containing eGFP-1; after transfection for 36 hours, cells were collected. , using flow cytometry analysis.
含有供体片段的质粒为将供体片段(核苷酸序列为序列8)***T3(全式金公司产品)载体上得到的载体。 The plasmid containing the donor fragment is a vector obtained by inserting a donor fragment (nucleotide sequence is sequence 8) into a T3 (full-scale product) carrier.
以PCS2-I-SceI载体与含有供体片段的质粒共转染为对照1；Co-transfected with PCS2-I-SceI vector and plasmid containing donor fragment as control 1;
以含有供体片段的质粒单独转染为对照2；The plasmid containing the donor fragment was separately transfected into control 2;
以PCS2-TALEN载体与含有供体片段的质粒共转染为对照3；Co-transfected with the PCS2-TALEN vector and the plasmid containing the donor fragment as control 3;
PCS2-TALEN载体为将序列表中序列5所示的TALEN编码基因***PCS2载体的NcoI和XbaI酶切位点得到的载体。The PCS2-TALEN vector is a vector obtained by inserting the TALEN encoding gene shown in SEQ ID NO: 5 into the NcoI and XbaI cleavage sites of the PCS2 vector.
结果如图6所示，与转染PCS2-I-SceI载体的转基因细胞相比，转染PCS2-TALE-I-SceI载体的转基因细胞(TALE-ISVBII)中GFP阳性细胞率为0.21％；转染PCS2-I-SceI载体的转基因细胞(ISVBII)中GFP阳性细胞率为0.10％；转染PCS2-TALEN载体的转基因细胞(TALEN)中GFP阳性细胞率为0.22％；NA中GFP阳性细胞率为0，可以看出，TALE-ISVBII表达TALE-ISceI蛋白能够显著提高修复效率为0.21％，ISVBII表达的I-SceI蛋白修复效率为0.1％。The results are shown in Fig. 6. Compared with transfected cells transfected with PCS2-I-SceI vector, the transfection rate of transfected cells (TALE-ISVBII) transfected with PCS2-TALE-I-SceI vector was 0.21%. The GFP positive cell rate in transgenic cells (ISVBII) infected with PCS2-I-SceI vector was 0.10%; the GFP positive cell rate in transgenic cells (TALEN) transfected with PCS2-TALEN vector was 0.22%; 0, it can be seen that TALE-ISVBII expression of TALE-ISceI protein can significantly improve the repair efficiency of 0.21%, ISVBII expression of I-SceI protein repair efficiency of 0.1%.
三、融合蛋白TALE-I-SceI对细胞毒性低Third, the fusion protein TALE-I-SceI has low cytotoxicity
γ-H2AX和53BP1蛋白都是用来检测细胞产生DSB的生物标记，为细胞毒性的表征蛋白，通过对这两个生物标记实验的检测。γ-H2AX和53BP1免疫荧光染色多显示TALEN的基因毒性高。Both γ-H2AX and 53BP1 proteins are used to detect the biomarkers that produce DSB in cells, and are characterization proteins for cytotoxicity by detecting these two biomarker experiments. Immunofluorescence staining of γ-H2AX and 53BP1 showed that the gene toxicity of TALEN was high.
实验方法如下：The experimental method is as follows:
1将1X106个293细胞铺在6孔板中。过24h时候，在细胞中分别转染1ug的由实施例1制备的PCS2-I-SceI、PCS2-TALEN和PCS2-TALE-I-SceI。1 1X10 6 293 cells were plated in 6-well plates. At 24 h, 1 ug of PCS2-I-SceI, PCS2-TALEN and PCS2-TALE-I-SceI prepared in Example 1 were transfected into cells, respectively.
2、转染后培养36h，将6孔板的细胞做免疫荧光染色实验，实验的步骤可以简要如下：4％甲醛固定15min,0.1％TritonX-100透膜10min,10％FBS封闭1h,抗γ-H2AX和53BP1蛋白的抗体孵育1h,PBS洗3遍5min,二抗孵育1h,PBS洗5min,DAPI染色15min,PBS洗3遍5min,将所得的细胞用于显微镜观察或者流式分析。2. After transfection, the cells were cultured for 36 hours. The cells of the 6-well plate were subjected to immunofluorescence staining. The experimental procedure can be briefly as follows: 4% formaldehyde for 15 min, 0.1% Triton X-100 for 10 min, 10% FBS for 1 h, anti-γ -H2AX and 53BP1 protein antibodies were incubated for 1 h, washed with PBS for 3 min for 5 min, secondary antibody for 1 h, washed with PBS for 5 min, DAPI stained for 15 min, washed with PBS for 3 min for 5 min, and the resulting cells were used for microscopic observation or flow analysis.
结果如图7所示，a为γ-H2AX结果，b为53BP1结果，可以看出，与转染PCS2-I-SceI的转基因细胞相比，转染PCS2-TALE-I-SceI的转基因细胞能够显著降低细胞毒性，体现在γ-H2AX和53BP1的蛋白水平表达显著降低。The results are shown in Figure 7. A is the result of γ-H2AX and b is the result of 53BP1. It can be seen that the transfected cells transfected with PCS2-TALE-I-SceI can be compared with the transfected cells transfected with PCS2-I-SceI. Significantly reduced cytotoxicity, reflected in a significant decrease in the expression of γ-H2AX and 53BP1 protein levels.
与转染PCS2-I-SceI的转基因细胞(ISVB2)和转染PCS2-TALEN的转基因细胞(TALEN)相比，转染PCS2-TALE-I-SceI的转基因细胞 (TALE-ISVB2)中γ-H2AX和53BP1的蛋白水平表达显著降低，可以看出，融合蛋白TALE-I-SceI对细胞毒性低。Transgenic cells transfected with PCS2-TALE-I-SceI compared to transfected cells transfected with PCS2-I-SceI (ISVB2) and transfected cells transfected with PCS2-TALEN (TALEN) The protein level expression of γ-H2AX and 53BP1 was significantly decreased in (TALE-ISVB2), and it can be seen that the fusion protein TALE-I-SceI has low cytotoxicity.
上述结果显示了新型基因打靶技术TALE-ISceI融合酶在临床应用上的优势，展示应用TALE-Iscel融合酶根治β-地中海贫血病的可行性。The above results show the advantages of the novel gene targeting technology TALE-ISceI fusion enzyme in clinical application, and demonstrate the feasibility of using TALE-Iscel fusion enzyme to cure β-thalassemia.
工业应用Industrial application
本发明的实验证明，本发明利用归巢内切酶I-SceI的低基因毒性和TALEN核酸酶的高效打靶优势，构建融合型内切酶TALE-ISceI，以β-地中海贫血疾病患者造血干细胞为模型，对其突变基因进行修复。该酶在体外β-地中海贫血疾病患者造血干细胞中发挥作用，有望在临床中得到广泛应用。同时，可以通过筛选从而使该酶识别并修复更多的核酸片段，将其推广应用到其他遗传型疾病治疗中。The experiments of the present invention prove that the present invention utilizes the low genotoxicity of homing endonuclease I-SceI and the high-efficiency targeting advantage of TALEN nuclease to construct a fusion endonuclease TALE-ISceI, which is a hematopoietic stem cell of a patient with β-thalassemia disease. Model to repair its mutant genes. The enzyme plays a role in hematopoietic stem cells of patients with β-thalassemia in vitro and is expected to be widely used in clinical practice. At the same time, the enzyme can be screened so that the enzyme recognizes and repairs more nucleic acid fragments and promotes them to other genetic diseases.
利用此新型基因打靶技术，目的是在体外修复β-地中海贫血患者的造血干细胞所携带基因的突变，并将修复过的造血干细胞分化为能正常表达β珠蛋白的红细胞。在造血干细胞上直接进行基因修复具有如下优势：避免了iPS转基因的不稳定性及iPS细胞的可能致癌性，同时避免移殖细胞的免疫排斥性等。因此，此工程酶的获得为β地中海贫血患者的彻底根治提供实践依据。 The novel gene targeting technology is used to repair mutations in genes carried by hematopoietic stem cells of patients with β-thalassemia in vitro, and to differentiate the repaired hematopoietic stem cells into red blood cells capable of normally expressing β-globin. Direct gene repair on hematopoietic stem cells has the following advantages: avoidance of iPS transgene instability and possible carcinogenicity of iPS cells, while avoiding immunological rejection of transplanted cells. Therefore, the acquisition of this engineered enzyme provides a practical basis for the radical cure of patients with beta thalassemia.

Claims (18)

  1. 一种蛋白质，为如下1)或2)：A protein as follows 1) or 2):
    1)所示的融合蛋白，为将TALE蛋白FokI替换成改造后I-SceI蛋白得到的蛋白；1) The fusion protein shown is a protein obtained by replacing the TALE protein FokI with the modified I-SceI protein;
    所述改造后I-SceI蛋白的氨基酸序列为序列表中序列2自N’末端第706-940位氨基酸；The amino acid sequence of the engineered I-SceI protein is amino acid sequence 706-940 of the sequence 2 in the sequence listing;
    2)所示的蛋白的氨基酸序列为序列表中序列2自N’末端第706-940位氨基酸。2) The amino acid sequence of the protein shown is the amino acid sequence 706-940 from the N' end of the sequence 2 in the sequence listing.
  2. 根据权利要求1所述的融合蛋白，其特征在于：The fusion protein of claim 1 wherein:
    所述TALE蛋白的氨基酸序列为序列表中序列4；The amino acid sequence of the TALE protein is the sequence 4 in the sequence listing;
    1)所示的融合蛋白的氨基酸序列为序列表中序列2。1) The amino acid sequence of the fusion protein shown is Sequence 2 in the Sequence Listing.
  3. 编码权利要求1或2所述的蛋白的核酸分子。A nucleic acid molecule encoding the protein of claim 1 or 2.
  4. 根据权利要求3所述的核酸分子，其特征在于：所述核酸分子为如下1)-4)中任一所述的DNA分子：The nucleic acid molecule according to claim 3, wherein the nucleic acid molecule is a DNA molecule according to any one of the following 1) to 4):
    1)编码区为序列表中序列1所示的DNA分子；1) the coding region is the DNA molecule shown in SEQ ID NO: 1 in the sequence listing;
    2)编码区为序列表中序列1第2116-2823位核苷酸所示的DNA分子；2) the coding region is a DNA molecule represented by nucleotides 2116-2823 of SEQ ID NO: 1 in the sequence listing;
    3)在严格条件下与1)或2)杂交且编码具有相同功能蛋白的DNA分子；3) hybridizing to 1) or 2) under stringent conditions and encoding a DNA molecule having the same functional protein;
    4)与1)或2)具有90％以上同源性且编码具有相同功能蛋白的DNA分子。4) A DNA molecule having more than 90% homology with 1) or 2) and encoding a protein having the same function.
  5. 含有权利要求3或4所述DNA分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。An expression cassette, recombinant vector, recombinant strain, transgenic cell line or recombinant strain comprising the DNA molecule of claim 3 or 4.
  6. 根据权利要求5所述的重组载体，其特征在于：所述重组载体为将权利要求3或4所述核酸分子***表达载体得到的重组载体。The recombinant vector according to claim 5, wherein the recombinant vector is a recombinant vector obtained by inserting the nucleic acid molecule of claim 3 or 4 into an expression vector.
  7. 根据权利要求5所述的重组菌，其特征在于：所述重组菌为将所述重组载体导入目的菌中得到的重组菌。The recombinant bacterium according to claim 5, wherein the recombinant bacterium is a recombinant bacterium obtained by introducing the recombinant vector into a target bacterium.
  8. 权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在切割目的基因和/或修复目的基因中的应用。The protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, the expression cassette comprising the nucleic acid molecule of claim 3 or 4, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain for cleavage of a gene of interest And/or repairing applications in the gene of interest.
  9. 权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含 有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在提高切割目的基因效率、提高修复目的基因效率或降低细胞毒性中的应用。The protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, The use of an expression cassette, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain having the nucleic acid molecule according to claim 3 or 4 for improving the efficiency of cleavage of a gene of interest, improving the efficiency of repairing a gene of interest or reducing cytotoxicity.
  10. 根据权利要求8或9所述的应用，其特征在于：所述目的基因为β地中海贫血症beta-globin基因。The use according to claim 8 or 9, wherein the gene of interest is a beta thalassemia beta-globin gene.
  11. 权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在制备治疗或辅助治疗β-地中海贫血病产品中的应用；The protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, the expression cassette comprising the nucleic acid molecule of claim 3 or 4, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain in the preparation of a treatment or Auxiliary treatment of applications in beta-thalassemia products;
  12. 权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌在制备体外修复β-地中海贫血患者的造血干细胞所携带基因的突变产品中的应用；所述基因具体为β地中海贫血症beta-globin基因。The protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, the expression cassette comprising the nucleic acid molecule of claim 3 or 4, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain for in vitro repair The use of a mutant product of a gene carried by a hematopoietic stem cell of a β-thalassemia patient; the gene is specifically a beta-globin gene of beta thalassemia.
  13. 一种切割目的基因和/或修复目的基因的试剂盒，包括权利要求1或2所述的蛋白、权利要求2或3所述核酸分子或含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。A kit for cleavage of a gene of interest and/or a gene of interest, comprising the protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, or an expression cassette comprising the nucleic acid molecule of claim 3 or Recombinant vector, recombinant strain, transgenic cell line or recombinant strain.
  14. 一种提高切割目的基因效率、提高修复目的基因效率或降低细胞毒性的试剂盒，包括权利要求1或2所述的蛋白、权利要求2或3所述核酸分子或含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。A kit for improving the efficiency of cleavage of a gene of interest, for improving the efficiency of repairing a gene of interest, or for reducing cytotoxicity, comprising the protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3 or the method of claim 3 or 4 An expression cassette, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain of a nucleic acid molecule.
  15. 一种体外修复β-地中海贫血患者的造血干细胞所携带基因的突变产品，其活性成分为权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。A mutant product for repairing a gene carried by a hematopoietic stem cell of a patient with β-thalassemia, wherein the active ingredient is the protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, or the method of claim 3 or 4 An expression cassette, a recombinant vector, a recombinant strain, a transgenic cell line or a recombinant strain of the nucleic acid molecule.
  16. 根据权利要求15所述的产品，其特征在于：所述基因为β地中海贫血症beta-globin基因；The product according to claim 15, wherein said gene is a β-thalassemia beta-globin gene;
    所述产品为药物或试剂盒。The product is a drug or kit.
  17. 一种治疗或辅助治疗β-地中海贫血病产品，其活性成分为权利要求1或2所述的蛋白、权利要求2或3所述核酸分子、含有权利要求3或4所述核酸分子的表达盒、重组载体、重组菌、转基因细胞系或重组菌。 A therapeutic or adjunctive treatment of a β-thalassemia product, wherein the active ingredient is the protein of claim 1 or 2, the nucleic acid molecule of claim 2 or 3, and the expression cassette comprising the nucleic acid molecule of claim 3 or 4. , recombinant vector, recombinant strain, transgenic cell line or recombinant strain.
  18. 根据权利要求17所述的产品，其特征在于：所述产品为药物或试剂盒。 The product of claim 17 wherein the product is a drug or kit.
PCT/CN2015/094901 2014-12-04 2015-11-18 Chimeric nuclease for specifically recognizing and repairing β thalassemia beta-globin gene WO2016086768A1 (en)

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