WO2021083183A1 - 一种造血干细胞hbb基因修复的方法及产品 - Google Patents

一种造血干细胞hbb基因修复的方法及产品 Download PDF

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WO2021083183A1
WO2021083183A1 PCT/CN2020/124303 CN2020124303W WO2021083183A1 WO 2021083183 A1 WO2021083183 A1 WO 2021083183A1 CN 2020124303 W CN2020124303 W CN 2020124303W WO 2021083183 A1 WO2021083183 A1 WO 2021083183A1
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sgrna
gene
cells
codon
hematopoietic stem
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吴宇轩
杨菲
席在喜
李大力
刘明耀
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上海邦耀生物科技有限公司
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Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a method and product for HBB gene repair of hematopoietic stem cells.
  • the system consists of Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (CAS) genes.
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • CAS CRISPR-associated genes.
  • the immune interference process of the CRISPR system mainly includes three stages: adaptation, expression and interference.
  • the adaptation stage the CRISPR system integrates short fragments of DNA from the phage or plasmid into the leader sequence and the first repeat sequence. Each integration is accompanied by the replication of the repeat sequence to form a new repeat-spacer sequence unit.
  • the CRISPR locus will be transcribed into a CRISPR RNA (crRNA) precursor (pre-crRNA), which will be in the presence of Cas protein and trans-encoded small RNA (tracrRNA)
  • crRNA CRISPR RNA
  • pre-crRNA CRISPR RNA
  • tracrRNA trans-encoded small RNA
  • the repetitive sequences are further processed into small crRNAs.
  • Mature crRNA and Cas protein form a Cas/crRNA complex.
  • crRNA guides the Cas/crRNA complex to find the target through its complementary region to the target sequence, and causes double-stranded DNA breaks at the target site through the nuclease activity of the Cas protein at the target site, thereby causing the target DNA to lose the original There are functions.
  • the 3 bases immediately adjacent to the 3'end of the target must be in the form of 5'-NGG-3' to form the PAM (protospacer advanced motif) structure required for the Cas/crRNA complex to
  • the CRISPR system is divided into three families, type I, II, and III.
  • the type II system only requires Cas9 protein to process pre-crRNA into mature crRNA that binds to tracrRNA with the assistance of tracrRNA. It has been found that by artificially constructing a single-stranded chimera guide RNA (guide RNA, also known as sgRNA) that mimics the crRNA:tracrRNA complex, the Cas9 protein can effectively mediate the recognition and cleavage of the target by the Cas9 protein, so as to be used in target species.
  • guide RNA also known as sgRNA
  • the CRISPR system provides broad prospects for modifying target DNA.
  • Beta-thalassemia is a common genetic disease that causes abnormal hemoglobin in adults due to defects in the beta-globin gene (HBB gene).
  • HBB gene beta-globin gene
  • the codon (Codon) 41/42 (-TCTT) genotype is the most common type of "thalassaemia” in my country.
  • the pathogenesis is that the HBB gene codon 41/42 (-TCTT) frameshift mutation causes amino acid encoding Abnormal and formation of stop codons leads to the premature termination of protein translation, and ultimately the loss of the function of the HBB gene.
  • the ideal gene therapy method is to repair or destroy the traditional thalassaemia mutations in the DNA of the patient’s hematopoietic stem cells, restore gene function, and permanently produce wild-type adult ⁇ -globin under the action of endogenous transcription control factors, thus normal Differentiate into erythroid cells.
  • the repair method of DNA sequence after gene editing is mainly by non-homologous end joining (NHEJ) repair, and the proportion of homologous recombination-mediated repair (Homology directed repair, HDR) is low.
  • NHEJ non-homologous end joining
  • HDR homologous recombination-mediated repair
  • the repair of point mutations requires high-efficiency HDR efficiency.
  • the present invention provides a method and product for HBB gene repair of hematopoietic stem cells.
  • the method uses the CRISPR-Cas9 system to repair the abnormal amino acid coding caused by codon 41/42 (-TCTT).
  • the inventors conducted in-depth research on the abnormal amino acid encoding caused by the frameshift mutation of codon 41/42 (-TCTT) and found that the mutation of codon 41/42 (-TCTT) caused the deletion of the 42nd codon of the gene encoding HBB.
  • TCTT thus causes the disorder of amino acid coding, and forms a stop codon not far after the deletion site, which makes the protein translation stop prematurely, leading to the loss of gene function.
  • the CRISPR-Cas9 system cuts the target DNA site to produce double-strand breaks, and there will be a high probability of frameshift mutations, where the number of bases after the indel at the pathogenic site changes if it is 3n+4 (n is an integer) , That is, it is possible to repair the amino acid coding disorder in the patient's genotype.
  • the strategy adopted by the inventors greatly increases the probability of homologous recombination during DNA repair, and can increase the proportion of ⁇ -globin mRNA produced by normal transcription in the genomic region and translated into it. Normal ⁇ -globin.
  • the invention aims at the frameshift mutation of the pathogenic site, cuts the target DNA and introduces the donor to achieve high-efficiency HDR.
  • the invention only need to transplant the autologous hematopoietic stem cells that have been repaired by the gene-edited method to greatly improve the HDR method and transplant them back into the body to achieve the goal of cure.
  • CRISPR-Cas9 can be used to target the target DNA to cause a double-strand break and simultaneously introduce a long-chain donor (ssODN).
  • ssODN long-chain donor
  • the designed sgRNA targeting sequence is 1 bp before the codon 41/42 (-TCTT) site.
  • the CRISPR-Cas system refers to a CRISPR-Cas system suitable for artificial modification, a nuclease system derived from Archaea type II (CRISPR)-CRISPR-associated protein (Cas) system, which is similar to ZFN and TALEN Compared with, the system is simpler and more convenient to operate.
  • CRISPR nuclease system derived from Archaea type II (CRISPR)-CRISPR-associated protein (Cas) system
  • RNA-guided endonucleases RNA-guided endonucleases
  • RGENs are composed of chimeric guide RNA and Cas9 protein.
  • the former is the fusion of CRISPR RNAs (crRNAs) and trans-activating crRNA (tracrRNA) in the naturally-occurring type II CRISPR-Cas system into a single-stranded guide RNA (sgRNA). ), which binds to the Cas9 protein and guides the latter to specifically cleavage the target DNA sequence.
  • the cleavage will form a double strand break (DSB).
  • This damage is connected through error-prone non-homologous ends (Non- Homologous end joining (NHEJ) repair can efficiently repair the mutation of the target gene, and complete the repair of the diseased site by homologous recombination.
  • NHEJ Non- Homologous end joining
  • the Cas9 may be selected from Cas9 derived from Streptococcus pyogenes, Staphylococcus aureus or N. meningitidis.
  • the Cas9 can be selected from wild-type Cas9 or mutant Cas9; the mutant Cas9 does not cause loss of Cas9 cleavage activity and targeting activity.
  • Cas enzymes can be used to replace Cas9.
  • sgRNA is used to cut the target sequence
  • the target sequence of the sgRNA is CCCCAAAGGACTCAACCTC (SEQ ID No. 1), CCCCAAAGGACTCAACCTCT (SEQ ID No. 2), GGACTCAACCTCTGGGTCCA (SEQ ID No. 3), GACTCAACCTCTGGGTCCAA (SEQ ID No. 4), GACCCAGAGGTTGAGTCCTT (SEQ ID No. 5), ACCCAGAGGTTGAGTCCTTT (SEQ ID No. 6), CCCAGAGGTTGAGTCCTTTG (SEQ ID No.
  • sgRNA can guide Cas9 at codon 41/42 (-TCTT)
  • the DNA is cut 1 base upstream of the point to cause a double-strand break, and at the same time, a normal donor is introduced for homologous recombination, thereby repairing the disorder of amino acid encoding with the greatest probability.
  • a normal genotype long chain donor (ssODN) is used for homologous recombination, and the ssODN sequence is TCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG (SEQ ID No. 8), and ssODN is introduced as a healthy donor for homologous recombination.
  • CRISPR-Cas9 to target the target DNA to cause a double-strand break and introduce the donor ssODN at the same time, homologous recombination can be introduced during the DNA repair process, which further enables the genomic region to be transcribed to produce normal HBB gene mRNA, and translated to produce normal ⁇ - Globin.
  • the present invention provides a method for repairing HBB ( ⁇ -globin gene) codon frameshift mutations in cells, including the steps of introducing nuclease and sgRNA into the cell and performing gene editing on the HBB gene.
  • the sgRNA guides The nuclease cuts the HBB gene and forms a break site;
  • the codon frameshift mutation is a frameshift mutation caused by the mutation of codon 41/42 (-TCTT);
  • the sgRNA targets the target sequence of the HBB gene Including 1 bp upstream of codon 41/42 (-TCTT) site;
  • the targeting sequence of the sgRNA targeting the HBB gene includes the sequence shown in any one of SEQ ID No. 1-7; more preferably, the targeting sequence of the sgRNA targeting the HBB gene includes SEQ ID No. 1-7. 1 shows the sequence.
  • the nuclease is selected from one or more of Cas9, Cas3, Cas8a, Cas8b, Cas10d, Cse1, Csy1, Csn2, Cas4, Cas10, Csm2, Cmr5, Fok1, Cpf1; preferably, the nuclease is Cas9; More preferably, the Cas9 is selected from Cas9 derived from Streptococcus pneumoniae, Streptococcus pyogenes or Streptococcus thermophilus.
  • the sgRNA also includes chemical modification of the base; preferably, the chemical modification is one or more of methylation modification, methoxy modification, fluorination modification or thio modification.
  • the sgRNA includes chemical modification of bases.
  • the sgRNA includes a chemical modification of any one or several bases from the 1 to n positions of the 5'end, and/or any one of the 1 to n bases from the 3'end Or chemical modification of any number of bases; the n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • the sgRNA includes one, two, three, four, or five base chemical modifications at the 5'end, and/or one, two, three, four, or five base chemical modifications at the 3'end. Retouch.
  • Bases, bases 1-4, and bases 1-5 are chemically modified; and/or the bases at position 1, base 2, and base 3 at the 3'end of sgRNA , Base 4, base 5 or base 1-2, base 1-3, base 1-4, base 1-5 are chemically modified.
  • the chemical modification is one or more of methylation modification, fluorination modification or thio modification.
  • the method also includes the steps of providing a donor repair template and introducing the donor repair template into the cell; preferably, the donor repair template includes the normal sequence corresponding to the HBB codon 41/42 (-TCTT).
  • the sequence of the donor repair template is shown in SEQ ID No. 8.
  • the donor repair template is purified by hPAGE.
  • the cells are hematopoietic stem cells, preferably, the cells are CD34 + hematopoietic stem progenitor cells.
  • the method of introducing the nuclease, sgRNA or donor repair template into the cell includes: vector transformation, transfection, heat shock, electroporation, transduction, gene gun, microinjection; preferably, electroporation is used. More preferably, the nuclease and sgRNA are formed into a complex, or the nuclease, sgRNA and the donor repair template are formed into a complex, and then the complex is introduced into the cell by electroporation.
  • the electrotransformation method is used to introduce a complex containing Cas9, sgRNA and a donor repair template into the cell.
  • the molar ratio of Cas9 and sgRNA is 1:(0.5-3), preferably, 1:(1-2), more preferably 1:1, and preferably, the amount of donor repair template introduced is 100 ⁇ M.
  • the Cas9 and sgRNA form a complex by incubation; preferably, the temperature of the incubation is 20-50°C, preferably, 25-37°C; preferably, the time of the incubation is 2-30 Minutes, preferably 5-20 minutes.
  • the dosage ratio of the complex containing Cas9 and sgRNA to the cells is 20-100 ⁇ g complex: (1 ⁇ 10 2 -1 ⁇ 10 6 cells), preferably, 30 ⁇ g complex: (1 ⁇ 10 3 -1 ⁇ 10 5 ) cells.
  • the electrotransformed cells are cultured in the CD34 + EDM-1 differentiation system for 7 days, and the genomic DNA of the cells obtained in the above steps is extracted for genotype identification to determine the mutation efficiency; after the mutation is determined, the EDM-2 stage differentiation is performed for 4 days , EMD-3 stage differentiation for 7 days, after differentiation, RNA was extracted, reverse transcribed into cDNA, and qPCR was used to detect the mRNA of HBB gene.
  • the present invention protects gene-edited cells prepared by any of the methods described above.
  • the cells are hematopoietic stem cells, preferably, the cells are CD34 + hematopoietic stem progenitor cells; more preferably, the cells are isolated cells.
  • the present invention provides a sgRNA for repairing an abnormal amino acid encoding caused by a frameshift mutation of the HBB codon.
  • the frameshift mutation of the codon is a shift caused by the mutation of codon 41/42 (-TCTT).
  • Code mutation; the targeting sequence of the sgRNA is shown in SEQ ID No. 1-7; preferably, the targeting sequence of the sgRNA is shown in SEQ ID No. 1.
  • the present invention protects the application of any one of the above-mentioned sgRNAs or the cells in the preparation of products for treating and/or preventing ⁇ -thalassemia.
  • the present invention designs sgRNA for the target region of codon 41/42 (-TCTT) of HBB gene, which provides the possibility for more precise and flexible editing on the genome.
  • the introduction of the above-mentioned sgRNA and Cas9 protein into ⁇ -thalassaemia codon 41/42 (-TCTT) hematopoietic stem cells can efficiently cut the pathogenic site through DNA double-strand break (DSB) and introduce exogenous normal donors.
  • the homologous recombination of the human body can repair the frameshift of the target gene as much as possible, quickly and efficiently restore the expression of the HBB gene, and greatly increase the expression of ⁇ -globin in patients with ⁇ -thalassemia.
  • the repairing efficiency of the present invention can reach up to 24%, which is significantly higher than that achieved by using ZFN and TALEN, and can efficiently modify autologous hematopoietic stem cells to balance the hematopoietic system for a long time, greatly saving experimental time and manpower and material resources.
  • Figure 1 is a schematic diagram of the working principle of the CRISPR/Cas9 system.
  • Figure 2 is a schematic diagram of ⁇ -thalassaemia codon 41/42 (-TCTT) frameshift mutation sites and sgRNAs.
  • Figure 3 shows the efficiency of different sgRNAs combined with the exogenous template ssODN to repair the HBB gene.
  • Figure 4 is a schematic diagram of Sanger sequencing results.
  • the upper picture is the blank control group 1; the middle picture is the blank control group 2; the lower picture is the experimental group (electrotransformed sgRNA+ssODN).
  • Figure 5 is a schematic diagram of globin qPCR after repair and differentiation of the pathogenic site of the patient's hematopoietic stem cells.
  • Figure 6 shows the analysis of the enucleation rate when the patient's hematopoietic stem cells are edited and differentiated into red blood cells. The results show that the edited cells have a higher enucleation rate.
  • Figure 7 shows the cell volume analysis of the patient's hematopoietic stem cells after editing and differentiation into red blood cells at the pathogenic site. The results show that the edited cells have a larger volume.
  • Figure 8 shows that the edited hematopoietic stem cells successfully homing four months after transplantation of immunodeficient mice, and have a homing efficiency similar to that of unedited cells.
  • Figure 9 shows that the edited hematopoietic stem cells can be successfully differentiated into various blood cells including B cells and Myeloid in the bone marrow of mice four months after transplantation of immunodeficient mice.
  • Figure 10 is an analysis of the repair efficiency of human-derived cells in transplanted mouse bone marrow.
  • I represents CD34-positive cells before transplantation
  • E represents human-derived cells in mouse bone marrow after transplantation.
  • the present invention uses CRISPR-Cas9 gene editing technology to target and destroy codons 41/42 (-TCTT) of abnormal mutation sites in ⁇ -thalassemia, and construct a system that can recognize and guide Cas9 protein to target sequence of target gene.
  • Guide RNA sequence sgRNA is a method to target disease-causing target DNA and make it change. The method includes: introducing into defective hematopoietic stem cells the sgRNA-encoding nucleic acid and Cas9 protein that recognize the target gene, thereby comparing the target genomic DNA sequence Recognize and cut and introduce foreign donors for homologous recombination. Then, the cells are cultured in vitro to express the nuclease and cause a double-strand break in the target genomic DNA near the disease site, and then repair the DNA break site.
  • the repair methods include: (a) Non-homologous end junction repair: resulting in gene mutations (base insertions, deletions) being introduced into the target genome sequence. (b) Homologous recombination repair: the introduction of an exogenous donor sequence into the target genomic DNA sequence leads to changes in the endogenous target gene sequence.
  • ssODN is introduced as an exogenous donor.
  • Example 1 Efficient restoration of gene function by homologous recombination of hematopoietic stem cells at codon 41/42 of ⁇ -thalassaemia (-TCTT)
  • the patient is ⁇ -thalassemia double heterozygous, and the genotype is CD41-42/CD71-72.
  • sgRNA-1 CCCCAAAGGACTCAACCTC (SEQ ID No. 1),
  • sgRNA-2 CCCCAAAGGACTCAACCTCT (SEQ ID No. 2)
  • sgRNA-3 GGACTCAACCTCTGGGTCCA (SEQ ID No. 3),
  • sgRNA-4 GACTCAACCTCTGGGTCCAA (SEQ ID No. 4),
  • sgRNA-5 GACCCAGAGGTTGAGTCCTT (SEQ ID No. 5),
  • sgRNA-6 ACCCAGAGGTTGAGTCCTTT (SEQ ID No. 6),
  • sgRNA-7 CCCAGAGGTTGAGTCCTTTG (SEQ ID No. 7).
  • the homologous recombination donor is a normal genotype long chain donor (ssODN), and the sequence is TCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGT TCTT TGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG (SEQ ID No. 8), purified by hPAGE.
  • ssODN normal genotype long chain donor
  • the sgRNA-1-7 synthesized by chemical modification were mixed with Cas9 protein at a molar ratio of 1:1 and incubated for 10 minutes at room temperature to form a complex, and 1 ⁇ l of the donor prepared in step 3 at a concentration of 100 ⁇ M was added; 7 kinds of sgRNA and Cas9 were prepared Protein and homologous recombination donor complex; electrotransformed as follows:
  • electroporation kit proportions electroporation solution to take the patient-derived hematopoietic stem cells, the number of electrical switch does not exceed 10 5, after the cells were centrifuged using electroporation, resuspended, and then the above-described incubated sgRNA and Cas9 protein, homologues complexes recombinant for Gently mix (the ratio of the amount of sgRNA and Cas9 protein complex to hematopoietic stem cells is 30 ⁇ g complex: 1 ⁇ 10 5 cells), then transfer to the electroporation cup to avoid air bubbles during the operation; use the CD34 cell electroporation program EO -100 for electric transfer (Lonza-4D electric transfer instrument);
  • the electrotransformation target cells are hematopoietic stem cells from healthy humans, which are carried out according to the method of a. The difference is that the sgRNA, Cas9 protein and homologous recombination donor complex are replaced with equal volume of water.
  • the electrotransformation target cells are patient-derived hematopoietic stem cells, which are carried out according to the method of a, except that the sgRNA, Cas9 protein and homologous recombination donor complex are replaced with an equal volume of water.
  • the hematopoietic stem cells electrotransformed in step 4 were differentiated and cultured in vitro for 3-4 days, the appropriate amount of cells was collected, the genome was extracted, and the repair efficiency was detected by Sanger sequencing after PCR amplification (that is, the ratio of the number of cells whose HBB returned to normal to the number of cells tested) The cells continued to differentiate in EDM-1 medium to day 7, and the detection results are shown in Figure 3 and Figure 4.
  • the PCR amplification primer sequence is as follows:
  • 41/42-check-R CCACACTGATGCAATCATTCG (SEQ ID No. 10).
  • Figure 3 shows that the repair efficiency of the target site of patient-derived hematopoietic stem cells after electroporation is sufficiently high, all above 15%, of which sgRNA-1 is the highest, reaching 24%.
  • Figure 4 shows that the blank control group 1 in the upper figure (hematopoietic stem cells from healthy people without any RNP (ribonucleoprotein)) has normal sequencing peaks; the blank control group 2 in the middle figure (CD41-42/71-72 patients) Source hematopoietic stem cells, without introducing any RNP), the sequencing peak figure shows CD41-42 (-TCTT) heterozygous mutation; the experimental group in the figure below (electrotransformed sgRNA+ssODN), the sequencing peak figure shows the random number from the sgRNA cleavage site The bases of the lost and the spurious peaks are generated.
  • the differentiation can be continued in EDM-2 medium for 4 days. At this stage, the cells can be expanded in large quantities. After the EDM-2 differentiation stage is over, the cells are transferred to EDM-3 to continue. After differentiation for 7 days, after the differentiation, the hematopoietic stem cell RNA was extracted to obtain cDNA by reverse transcription, and q-PCR was used to analyze the changes in the HBB mRNA content of the hematopoietic stem cells after the pathogenic site mutation.
  • HBA-S GCCCTGGAGAGGATGTTC (SEQ ID No.11);
  • HBA-AS TTCTTGCCGTGGCCCTTA (SEQ ID No. 12);
  • HBB-S TGAGGAGAAGTCTGCCGTTAC (SEQ ID No. 13);
  • HBB-AS ACCACCAGCAGCCTGCCCA (SEQ ID No. 14).
  • the enucleation rate of the patient’s hematopoietic stem cells edited and differentiated into red blood cells was detected.
  • the results are shown in Figure 6.
  • the patients’ hematopoietic stem cells edited by sgRNA-1+ssODN had a higher enucleation rate (Y axis For the enucleation rate, the X axis is the hematopoietic stem cells from different patients).
  • the cell volume of the patient’s hematopoietic stem cells edited and differentiated into red blood cells was detected.
  • the results are shown in Figure 7.
  • the patient’s hematopoietic stem cells edited by sgRNA-1+ssODN in the experimental group have a larger volume (the Y axis is the cell volume).
  • X axis is hematopoietic stem cells from different patients).
  • the patient's hematopoietic stem cells edited by sgRNA-1+ssODN in the experimental group of Example 1 were transplanted into immunodeficient mice. After four months, they successfully homing, with similar homing efficiency to unedited cells (as shown in Figure 8,
  • the Y-axis shows the proportion of human-derived cells in mouse bone marrow); and can be successfully differentiated into various blood cells including B cells and myeloid cells in mouse bone marrow ( Figure 9); and the proportion of human-derived cells in mouse bone marrow
  • Figure 10 The results of repair efficiency analysis ( Figure 10) showed that a large part of the cells (Y axis) still possess the repaired HBB gene after transplantation.

Abstract

提供了一种造血干细胞HBB基因修复的方法及产品，所述方法利用CRISPR-Cas9基因编辑技术靶向敲除β-地中海贫血(地贫)中缺失位点密码子41/42(-TCTT)的技术，通过设计并合成能识别引导Cas9蛋白至目标基因靶序列的sgRNA，将其与Cas9蛋白混合电转导入β-地贫密码子41/42(-TCTT)造血干细胞中，同时引入同源重组供体高效修复该突变位点处氨基酸的正常编码功能，恢复β-珠蛋白基因的正常表达。

Description

一种造血干细胞HBB基因修复的方法及产品 技术领域
本发明涉及基因工程技术领域，具体涉及一种造血干细胞HBB基因修复的方法及产品。
背景技术
近年细菌和古细菌中一种用来抵御噬菌体和质粒等外源DNA片段入侵的获得性免疫机制得到了阐释。该系统由Clustered regularly interspaced short palindromic repeats(CRISPR)和CRISPR-associated(CAS)基因组成。CRISPR系统的免疫干扰过程主要包括3个阶段：适应、表达和干扰。适应阶段，CRISPR系统会将来自噬菌体或质粒的DNA短片段整合到前导序列和第一段重复序列之间，每一次整合都伴随着重复序列的复制，进而形成一个新的重复-间隔序列单元。表达阶段，CRISPR基因座会被转录成一段CRISPR RNA(crRNA)前体(pre-crRNA)，该前体在Cas蛋白和反式编码小RNA(trans-encoded small RNA，tracrRNA)的存在下会在重复序列处被进一步加工成小的crRNA。成熟的crRNA与Cas蛋白形成Cas/crRNA复合体。干扰阶段，crRNA通过其与靶序列互补的区域引导Cas/crRNA复合体寻找靶点，并在靶点位置通过Cas蛋白的核酸酶活性造成靶点位置的双链DNA断裂，从而使靶标DNA失去原有功能。其中与靶点3′端紧邻的3个碱基必须是5′-NGG-3′的形式，从而构成Cas/crRNA复合体识别靶点所需的PAM(protospacer adjacent  motif)结构。
CRISPR系统分为I，II，III型三个家族，其中II型系统仅需要Cas9蛋白即可在tracrRNA的协助下将pre-crRNA加工成与tracrRNA结合的成熟crRNA。人们发现通过人工构建模拟crRNA：tracrRNA复合体的单链嵌合体引导RNA(guide RNA，又称sgRNA)，即可有效的介导Cas9蛋白对靶点的识别和切割，从而为在目标物种中利用CRISPR系统对目标DNA进行修饰提供了广阔的前景。
β-地中海贫血是一种常见的由于β-珠蛋白基因(HBB基因)缺陷导致成人血红蛋白异常的遗传性疾病，我国“地贫”基因携带者约3000万人，涉及近3000万家庭、1亿人口，其中重型和中间型“地贫”患者约30万人。其中，密码子(Codon)41/42(-TCTT)基因型是我国“地贫”中最为多见的一种，发病机制为HBB基因密码子41/42(-TCTT)移码突变造成氨基酸编码异常并形成终止密码子导致蛋白翻译的提前终止，最终使得HBB基因的功能丧失。目前，中间型和重型患者需要长期输血和去铁治疗来维持生命，唯一根治方式为异体造血干胞移细植术，但主要实施障碍是我国血液资源的紧缺、异体造血干细胞配型困难及移植相关并发症等。其中，利用慢病毒载体进行基因治疗，表现出极大的潜力，但是半随机的载体整合方式，具致癌风险。同时慢病毒中的表达元件在造血干细胞长期归巢和自我更新的过程中会逐渐沉默，使得疗效下降，即有可能无法达到终身治愈的目的。另外，临床所需的高浓度、高质量的慢病毒对设备和技术的要求极高，因此成本很难降低。因此，并行的、更加安全、成本更低的临床方案 是非常有必要的。
理想的基因治疗方法，是修复或破坏患者造血干细胞DNA中传统的地贫突变，使之重新恢复基因功能，并在内源转录控制因子的作用下永久产生野生型成体β-珠蛋白，从而正常分化为红系细胞。DNA序列在基因编辑后的修复方式以非同源性末端接合(Non-homologous end joining，NHEJ)修复为主，同源重组介导的修复(Homology directed repair，HDR)所占的比例较低，而修复点突变需要高效的HDR效率。
发明内容
针对现有技术中存在的缺陷，本发明提供了一种造血干细胞HBB基因修复的方法及产品。所述方法利用CRISPR-Cas9系统对密码子41/42(-TCTT)导致的氨基酸编码异常进行修复。
发明人针对密码子41/42(-TCTT)移码突变导致的氨基酸编码异常进行了深入的研究，发现密码子41/42(-TCTT)的突变使得编码HBB基因的第42个密码子处缺失TCTT从而造成氨基酸编码的紊乱，并在缺失位点后不远处形成终止密码子使得蛋白翻译提前终止，导致基因功能的丧失。
理论上，CRISPR-Cas9系统切割目标DNA位点产生双链断裂，会出现大概率的移码突变，其中在致病位点处indel后碱基数的改变若为3n+4(n为整数)，即有可能修复患者基因型中的氨基酸编码紊乱，发明人采用的策略大大增加了DNA修复过程中同源重组的概率，可增加基因组区域正常转录产生的β-珠蛋白mRNA比例，并翻译产生正常的β-珠蛋白。
本发明针对致病位点的移码突变，靶向切割目标DNA并引入供体实现高效HDR。在临床上，只需将基因编辑后大幅提高HDR方式修复的自体造血干细胞移植回体内，便可达到治愈的目的。
在一个实施方式中，可以采用CRISPR-Cas9靶向目标DNA造成双链断裂并同时引入长链供体(ssODN)。
优选的，采用CRISPR-Cas9系统时，所设计的sgRNA的靶向序列在密码子41/42(-TCTT)位点前1bp。
本发明中，所述CRISPR-Cas系统是指适合被人工改造的CRISPR-Cas系统、源于古细菌II型(CRISPR)-CRISPR-associated protein(Cas)系统的核酸酶体系，与ZFN和TALEN相比，该体系更简单、操作更方便。
本发明采用RNA引导的核酸内切酶(RNA-guided endonucleases，RGENs)实现对目标基因序列的特异性切割。RGENs由嵌合型的引导RNA和Cas9蛋白组成，其中前者是将天然存在的II型CRISPR-Cas系统中的CRISPR RNAs(crRNAs)与trans-activating crRNA(tracrRNA)融合成一条单链引导RNA(sgRNA)，从而与Cas9蛋白结合并引导后者对目标DNA序列进行特异性的切割，切割将形成双链断裂(double strand break,DSB)，该损伤通过易错性的非同源末端连接(Non-homologous end joining，NHEJ)进行修复可高效修复目标基因的突变，以同源重组方式对致病位点处进行完全修复。
所述Cas9可以选自Streptococcus pyogenes、Staphylococcus aureus或N.meningitidis来源的Cas9。所述Cas9可以选自野生型的Cas9，也可以选自突变型的Cas9；所述突变型的Cas9不导致Cas9的切割活性和靶向活性丧失。
在其他的实施方式中，还可以采用其他的Cas酶替换Cas9。
在实施方式中，采用sgRNA进行目标序列的切割，所述sgRNA的靶向序列分别为CCCCAAAGGACTCAACCTC(SEQ ID No.1)，CCCCAAAGGACTCAACCTCT(SEQ ID No.2)，GGACTCAACCTCTGGGTCCA(SEQ ID No.3)， GACTCAACCTCTGGGTCCAA(SEQ ID No.4)，GACCCAGAGGTTGAGTCCTT(SEQ ID No.5)，ACCCAGAGGTTGAGTCCTTT(SEQ ID No.6)，CCCAGAGGTTGAGTCCTTTG(SEQ ID No.7)，sgRNA可以引导Cas9在密码子41/42(-TCTT)位点上游1个碱基处切割DNA造成双链断裂，同时引入正常供体进行同源重组，从而最大概率修复氨基酸编码的紊乱。
在一个实施方式中，采用正常基因型长链供体(ssODN)进行同源重组，所述ssODN序列为TCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGTTCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG(SEQ ID No.8)，ssODN作为健康供体引入同源重组。
采用CRISPR-Cas9靶向目标DNA造成双链断裂并同时引入供体ssODN，可在DNA修复过程中引入同源重组，进一步使得基因组区域转录产生正常的HBB基因的mRNA，并翻译产生正常的β-珠蛋白。
发明详述：
一方面，本发明提供了一种修复细胞中HBB(β-珠蛋白基因)密码子移码突变的方法，包括利用核酸酶和sgRNA引入细胞、对HBB基因进行基因编辑的步骤，所述sgRNA引导核酸酶对HBB基因进行切割并形成断裂位点；所述密码子移码突变为密码子41/42(-TCTT)的突变所导致的移码突变；所述sgRNA靶向HBB基因的靶向序列包括密码子41/42(-TCTT)位点的上游1bp；
优选的，所述sgRNA靶向HBB基因的靶向序列包含SEQ ID No.1-7中任一所示的序列；更优选的，所述sgRNA靶向HBB基因的靶向序列包含SEQ ID No.1所示的序列。
所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种；优选的，所述核酸酶为Cas9；更优选的，所述Cas9选自来源于肺炎链球菌、化脓性链球菌或嗜热链球菌的Cas9。
所述sgRNA还包括碱基的化学修饰；优选的，所述化学修饰为甲基化修饰、甲氧基修饰、氟化修饰或硫代修饰中的一种或任意几种。
在一个实施方式中，所述sgRNA包括碱基的化学修饰。在优选的实施方式中，所述sgRNA包括5’末端第1-n位碱基的任意一个或任意几个碱基的化学修饰，和/或3’末端第1至n位碱基的任意一个或任意几个碱基的化学修饰；所述n选自2、3、4、5、6、7、8、9或10。优选的，sgRNA包括5’末端一个、两个、三个、四个或五个碱基的化学修饰，和/或3’末端一个、两个、三个、四个或五个碱基的化学修饰。例如，在sgRNA的5’末端第1位碱基、第2位碱基、第3位碱基、第4位碱基、第5位碱基或第1-2位碱基、第1-3位碱基、第1-4位碱基、第1-5位碱基进行化学修饰；和/或，在sgRNA的3’末端第1位碱基、第2位碱基、第3位碱基、第4位碱基、第5位个碱基或第1-2位碱基、第1-3位碱基、第1-4位碱基、第1-5位碱基进行化学修饰。在优选的实施方式中，所述化学修饰为甲基化修饰、氟化修饰或硫代修饰中的一种或任意几种。
所述方法还包括提供供体修复模板以及将供体修复模板引入细胞的步骤；优选的，所述供体修复模板包括所述HBB密码子41/42(-TCTT)对应的正常序列。
在优选的实施方式中，所述供体修复模板的序列如SEQ ID No.8所示，优选的，所述供体修复模板采用hPAGE纯化。
在上述方法中，所述细胞为造血干细胞，优选的，所述细胞为CD34 +的造血干祖细胞。
在上述方法中，所述将核酸酶、sgRNA或供体修复模板引入细胞的方式包括：载体转化、转染、热休克、电穿孔、转导、基因枪、显微注射；优选的，采用电穿孔的方式；更优选的，将核酸酶和sgRNA形成复合物，或者将核酸酶、sgRNA和供体修复模板形成复合物，再将复合物通过电穿孔的方式引入到细胞中。
在优选的实施方式中，采用电转化的方法向细胞中引入包含Cas9和sgRNA及供体修复模板的复合物。
进一步的，所述Cas9和sgRNA的摩尔比为1:(0.5-3)，优选的，1:(1-2)，更优选1:1，优选的，供体修复模板的引入量为100μM。
进一步的，所述Cas9和sgRNA通过温育形成复合物；优选的，所述温育的温度为20-50℃，优选，25-37℃；优选的，所述温育的时间为2-30分钟，优选，5-20分钟。
进一步的，所述包含Cas9和sgRNA的复合物与细胞的用量比例为20-100μg复合物：(1×10 2-1×10 6个)细胞，优选的，为30μg复合物：(1×10 3-1×10 5个)细胞。
进一步的，将电转后的细胞在CD34 +EDM-1分化体系中培养7天，提取上述步骤所得细胞的基因组DNA进行基因型鉴定，确定突变效率；待确定突变后进行EDM-2阶段分化4天，EMD-3阶段分化7天，分化结束后提取RNA,反转录为cDNA，qPCR检测HBB基因的mRNA。
本发明保护以上任一所述的方法制备得到的基因编辑的细胞。进一步的，所述细胞为造血干细胞，优选的，所述细胞为CD34 +的造血干祖细胞；更优选的，所述细胞为离体细胞。
另一方面，本发明提供了一种用于修复由HBB密码子移码突变导致氨基酸编码异常的sgRNA，所述密码子移码突变为密码子41/42(-TCTT)的突变所导致的移码突变；所述sgRNA的靶向序列为SEQ ID No.1-7所示；优选的，所述sgRNA的靶向序列为SEQ ID No.1所示。
另一方面，本发明保护以上任一所述sgRNA或所述细胞在制备治疗和/或预防β地中海贫血症的产品中的应用。
有益效果：
本发明针对HBB基因的密码子41/42(-TCTT)的靶向区域设计了sgRNA，为在基因组上进行更为精确和灵活的编辑提供了可能。将上述sgRNA与Cas9蛋白引入β-地贫密码子41/42(-TCTT)的造血干细胞，能高效切割致病位点，通过DNA双链断裂(double strand break,DSB)以及引入外源正常供体进行同源重组能最大可能修复目标基因的移码，快速高效恢复HBB基因的表达，使β地贫病人的β-珠蛋白 表达得到了极大提高。本发明的修复效率最高可达24％，显著高于采用ZFN、TALEN所能达到的效率，且能够高效修饰自体造血干细胞持久平衡造血系统，大大节省实验时间以及人力物力的投入。
附图说明
此处所说明的附图用来提供对本申请的进一步理解，构成本申请的一部分，本申请的示意性实施例及其说明用于解释本申请，并不构成对本申请的不当限定。在附图中：
图1为CRISPR/Cas9系统作用原理示意图。
图2为β-地贫密码子41/42(-TCTT)移码突变位点及sgRNAs的示意图。
图3为不同sgRNAs结合外源模板ssODN修复HBB基因的效率。
图4为Sanger测序结果示意图。其中，上图是空白对照组1；中图是空白对照组2；下图是实验组(电转sgRNA+ssODN)。
图5为患者造血干细胞致病位点修复分化后珠蛋白qPCR示意图。
图6为患者造血干细胞致病位点编辑分化为红细胞时的去核率分析。结果显示编辑过后的细胞具有更高的去核率。
图7为患者造血干细胞致病位点编辑分化为红细胞后的细胞体积分析。结果显示编辑过后的细胞具有更大的体积。
图8为编辑过后的造血干细胞在移植免疫缺陷小鼠四个月后成功归巢，并且具有和未编辑细胞相似的归巢效率。
图9为编辑过后的造血干细胞在移植免疫缺陷小鼠四个月后可以在小鼠骨髓中成功分化为包括B细胞和Myeloid在内的各种血液细胞。
图10为被移植小鼠骨髓中人源细胞的修复效率分析。其中，I表示移植前的CD34阳性细胞，E表示移植后小鼠骨髓中的人源细胞。
具体实施方式
结合以下具体实施例和附图，对本发明作进一步的详细说明，本发明的保护内容不局限于以下实施例。在不背离发明构思的精神和范围下，本领域技术人员能够想到的变化和优点都被包括在本发明中，并且以所附的权利要求书为保护范围。实施本发明的过程、条件、试剂、实验方法等，除以下专门提及的内容之外，均为本领域的普遍知识和公知常识，本发明没有特别限制内容。如按照Sambrook等人，分子克隆，实验室手册(New York:ColdSpring Harbor Laboratory Press,1989)所记载，或按照厂商的建议条件。
如图1所示，本发明利用CRISPR-Cas9基因编辑技术靶向破坏β-地中海贫血中异常突变位点密码子41/42(-TCTT)，构建能识别并引导Cas9蛋白至目标基因靶序列的引导RNA序列(sgRNA)，是一种靶向致病目标DNA并使其发生改变的方法，该方法包括：向缺陷造血干细胞引入识别目标基因的sgRNA编码核酸及Cas9蛋白，从而对目标基因组DNA序列进行识别和切割并引入外源供体进行同源重组。然后，将细胞进行体外培养，使核酸酶表达并在致病位点附近的目标基因组DNA发生双链断裂，接着对该DNA断裂位点进行修复。
其中，修复方式包括：(a)非同源末端连接修复：导致基因突变(碱基插入、缺失)被引入到目的基因组序列中。(b)同源重组修复：使外源供体序列引入到目标基因组DNA序列中，导致内源目标基因序列的改变。本实施方式中，引入ssODN作为外源供体。
实施例1、在β-地贫密码子41/42(-TCTT)造血干细胞同源重组高效恢复基因功能
本实施例中患者为β-地贫双重杂合子，基因型为CD41-42/CD71-72。
1、sgRNA的设计
基于密码子41/42(-TCTT)致病位点前1bp处有合适的PAM靶向切割，在致病位点处设计多条sgRNA，各条sgRNA的靶向序列(图2)如下：
sgRNA-1：CCCCAAAGGACTCAACCTC(SEQ ID No.1)，
sgRNA-2：CCCCAAAGGACTCAACCTCT(SEQ ID No.2)，
sgRNA-3：GGACTCAACCTCTGGGTCCA(SEQ ID No.3)，
sgRNA-4：GACTCAACCTCTGGGTCCAA(SEQ ID No.4)，
sgRNA-5：GACCCAGAGGTTGAGTCCTT(SEQ ID No.5)，
sgRNA-6：ACCCAGAGGTTGAGTCCTTT(SEQ ID No.6)，
sgRNA-7：CCCAGAGGTTGAGTCCTTTG(SEQ ID No.7)。
2、sgRNA、Cas9蛋白的制备
3、同源重组供体的设计和制备
所述同源重组供体为正常基因型长链供体(ssODN)，序列为TCCCACCCTTAGGCTGCTGGTGGTCTACCCTTGGACCCAGAGGT TCTTTGAGTCCTTTGGGGATCTGTCCACTCCTGATGCTGTTATGGG(SEQ ID No.8)，采用hPAGE纯化。
4、sgRNA以及Cas9蛋白复合物的制备和电转
a实验组
将经由化学修饰合成的sgRNA-1～7分别与Cas9蛋白按摩尔比1:1混合室温孵育10min后形成复合物，加入1μl浓度为100μM的步骤3制备的供体；制备得到7种sgRNA、Cas9蛋白和同源重组供体复合物；分别按照如下方式电转：
按电转试剂盒比例混合电转液，取患者来源造血干细胞，电转数量不超过10 5个，将细胞离心后使用电转液重悬，再与上述孵育的sgRNA和Cas9蛋白、同源重组供体复合物轻轻混匀(sgRNA和Cas9蛋白的复合物与造血干细胞的用量之比为30μg复合物：1×10 5个细胞)后转移至电转杯，操作过程中避免产生气泡；使用CD34细胞电转程序EO-100进行电转(Lonza-4D电转仪)；
确认电转成功后待细胞室温静置孵育5min，重新离心去除Cas9蛋白和电转液，CD34 +EDM-1培养基重悬细胞后加入细胞培养板37℃分化培养，完成对患者缺陷型造血干细胞致病突变位点的破坏。
b空白对照组1(CK1)
电转靶细胞为健康人来源造血干细胞，按照a的方法进行，不同之处在于：将sgRNA、Cas9蛋白和同源重组供体复合物替换为等体 积的水。
c空白对照组2(CK2)
电转靶细胞为患者来源造血干细胞，按照a的方法进行，不同之处在于：将sgRNA、Cas9蛋白和同源重组供体复合物替换为等体积的水。
5、目标基因编辑的鉴定
(1)基因组DNA的突变鉴定
将步骤4电转后的造血干细胞体外分化培养3-4天后，收集适量细胞，提取基因组，PCR扩增后Sanger测序检测修复效率(即HBB恢复正常的细胞数量占被检细胞数量的比例)，剩余细胞继续在EDM-1培养基中分化至第7天，检测结果如图3和图4所示。
其中，PCR扩增引物序列如下：
41/42-check-F：GCTTCTGACACAACTGTGTTC(SEQ ID No.9)；
41/42-check-R：CCACACTGATGCAATCATTCG(SEQ ID No.10)。
图3显示，电转后患者来源造血干细胞目标位点的修复效率足够高，均在15％以上，其中sgRNA-1最高，达24％。
图4显示，上图的空白对照组1(健康人来源造血干细胞，未导入任何RNP(核糖核蛋白))，测序峰图正常；中图的空白对照组2(CD41-42/71-72患者来源造血干细胞，未导入任何RNP)，测序峰图显示CD41-42(-TCTT)杂合突变；下图的实验组(电转sgRNA+ssODN)，测序峰图显示从sgRNA切割位点处由于随机数目 的碱基丢失而产生杂峰。
(2)q-PCR分析致病位点突变后的造血干细胞中β珠蛋白含量变化
待Sanger测序后确定目标位点突变成功后即可在EDM-2培养基中继续分化4天，此阶段细胞可大量扩增，待EDM-2分化阶段结束，将细胞转移至EDM-3中继续分化7天，待分化结束后提取造血干细胞RNA反转录获得cDNA，q-PCR分析致病位点突变后的造血干细胞HBB mRNA含量变化。
其中，q-PCR特异检测HBA(内参)和HBB所用引物对序列如下：
HBA-S：GCCCTGGAGAGGATGTTC(SEQ ID No.11)；
HBA-AS：TTCTTGCCGTGGCCCTTA(SEQ ID No.12)；
HBB-S：TGAGGAGAAGTCTGCCGTTAC(SEQ ID No.13)；
HBB-AS：ACCACCAGCAGCCTGCCCA(SEQ ID No.14)。
结果：如图5所示，与CK1的健康人来源的造血干细胞相比，CK2的患者来源的造血干细胞的HBB与HBA的mRNA比值几近为零，而实验组中经sgRNA-1+ssODN编辑后的造血干细胞中，HBB与HBA的mRNA比值增加到了30％以上。此比例足以消除HBA水平过高导致的红细胞毒性，可以有效缓解地中海贫血症状。
(3)分化为红细胞时的去核率和细胞体积分析
检测患者造血干细胞致病位点编辑分化为红细胞时的去核率，结果如图6所示，实验组中经sgRNA-1+ssODN编辑过后的患者造血干 细胞具有更高的去核率(Y轴为去核率，X轴为不同患者来源的造血干细胞)。
检测患者造血干细胞致病位点编辑分化为红细胞后的细胞体积，结果如图7所示，实验组中经sgRNA-1+ssODN编辑过后的患者造血干细胞具有更大的体积(Y轴为细胞体积，X轴为不同患者来源的造血干细胞)。
实施例2、编辑过后的造血干细胞
将实施例1实验组中经sgRNA-1+ssODN编辑过后的患者造血干细胞移植免疫缺陷小鼠，四个月后成功归巢，具有和未编辑细胞相似的归巢效率(如图8所示，Y轴显示小鼠骨髓中人源细胞比例)；且可以在小鼠骨髓中成功分化为包括B细胞和髓细胞在内的各种血液细胞(图9)；且小鼠骨髓中人源细胞的修复效率分析结果(图10)显示，有很大一部分细胞(Y轴)在移植后仍然拥有被修复的HBB基因。
以上结果表明，经sgRNA-1+ssODN编辑过后，患者造血干细胞功能正常，并未受到影响，且成功修复了能长期归巢的造血干细胞中的HBB基因。
以上所述仅为本申请的实施例而已，并不用于限制本申请。对于本领域技术人员来说，本申请可以有各种更改和变化。凡在本申请的精神和原理之内所作的任何修改、等同替换、改进等，均应包含在本申请的权利要求范围之内。

Claims (10)

  1. 一种修复细胞中HBB(β-珠蛋白基因)密码子移码突变的方法，其特征在于，所述方法包括利用核酸酶和sgRNA引入细胞、对HBB基因进行基因编辑的步骤，所述sgRNA引导核酸酶对HBB基因进行切割并形成断裂位点；所述密码子移码突变为密码子41/42(-TCTT)的突变所导致的移码突变；所述sgRNA靶向HBB基因的靶向序列包括密码子41/42(-TCTT)位点的上游1bp；
    优选的，所述sgRNA靶向HBB基因的靶向序列包含SEQ ID No.1-7中任一所示的序列；更优选的，所述sgRNA靶向HBB基因的靶向序列包含SEQ ID No.1所示的序列。
  2. 根据权利要求1所述的方法，其特征在于，所述核酸酶选自Cas9、Cas3、Cas8a、Cas8b、Cas10d、Cse1、Csy1、Csn2、Cas4、Cas10、Csm2、Cmr5、Fok1、Cpf1中的一种或任意几种；优选的，所述核酸酶为Cas9；更优选的，所述Cas9选自来源于肺炎链球菌、化脓性链球菌或嗜热链球菌的Cas9。
  3. 根据权利要求1或2所述的方法，其特征在于，所述sgRNA还包括碱基的化学修饰；优选的，所述化学修饰为甲基化修饰、甲氧基修饰、氟化修饰或硫代修饰中的一种或任意几种。
  4. 根据权利要求1-3中任一所述的方法，其特征在于，所述方法还包括提供供体修复模板以及将供体修复模板引入细胞的步骤；优选的，所述供体修复模板包括所述HBB密码子41/42(-TCTT)对应的正常序列。
  5. 根据权利要求4所述的方法，其特征在于，所述供体修复模 板的序列如SEQ ID No.8所示，优选的，所述供体修复模板采用hPAGE纯化。
  6. 根据权利要求1-5中任一所述的方法，其特征在于，所述细胞为造血干细胞，优选的，所述细胞为CD34 +的造血干祖细胞。
  7. 根据权利要求1-6中任一所述的方法，其特征在于，所述将核酸酶、sgRNA或供体修复模板引入细胞的方式包括：载体转化、转染、热休克、电穿孔、转导、基因枪、显微注射；优选的，采用电穿孔的方式；更优选的，将核酸酶和sgRNA形成复合物，或者将核酸酶、sgRNA和供体修复模板形成复合物，再将复合物通过电穿孔的方式引入到细胞中。
  8. 权利要求1-7中任一所述的方法制备得到的基因编辑的细胞。
  9. 一种用于修复由HBB密码子移码突变导致氨基酸编码异常的sgRNA，所述密码子移码突变为密码子41/42(-TCTT)的突变所导致的移码突变；所述sgRNA的靶向序列为SEQ ID No.1-7所示；优选的，所述sgRNA的靶向序列为SEQ ID No.1所示。
  10. 权利要求9所述sgRNA或权利要求8所述细胞在制备治疗和/或预防β地中海贫血症的产品中的应用。
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