WO2020253753A1 - 一种内含子异常剪接的修复方法、产品和应用 - Google Patents

一种内含子异常剪接的修复方法、产品和应用 Download PDF

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WO2020253753A1
WO2020253753A1 PCT/CN2020/096676 CN2020096676W WO2020253753A1 WO 2020253753 A1 WO2020253753 A1 WO 2020253753A1 CN 2020096676 W CN2020096676 W CN 2020096676W WO 2020253753 A1 WO2020253753 A1 WO 2020253753A1
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sgrna
site
ivs2
seq
sequence
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French (fr)
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吴宇轩
杨菲
李大力
刘明耀
席在喜
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上海邦耀生物科技有限公司
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Priority claimed from CN201910543865.5A external-priority patent/CN112111528B/zh
Priority claimed from CN202010097964.8A external-priority patent/CN112391410B/zh
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Definitions

  • the invention relates to the technical field of genetic engineering, in particular to a repair method, product and application of abnormal splicing of introns.
  • the system is composed 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 further processed into small crRNAs at the repetitive sequence in the presence of Cas protein and tracrRNA.
  • crRNA CRISPR RNA
  • pre-crRNA CRISPR RNA
  • 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
  • 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 recognize the target.
  • PAM protospacer advanced motif
  • CRISPR Clustered regularly interspaced short palindromic repeats
  • type II system only requires Cas9 protein to be assisted by trans-encoded small RNA (tracrRNA)
  • tracrRNA trans-encoded small RNA
  • the pre-crRNA is processed into mature crRNA combined with tracrRNA. It has been found that by artificially constructing a single-stranded chimera guide RNA (guide RNA) 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 use the CRISPR system to target the target species. Modification of DNA offers broad prospects.
  • Beta-thalassemia is a common genetic disease that causes abnormal hemoglobin in adults due to defects in the beta-globin gene.
  • the IVS2-654 C>T genotype is one of the more common “thalassaemias” in my country.
  • the pathogenesis is the C>T mutation at base 654 in the second intron of the HBB gene, resulting in abnormalities.
  • the splicing site results in an extra 73 nt exons in the ⁇ -globin mRNA and premature termination of translation.
  • the original expression in the lentivirus will gradually be silent during the long-term homing and self-renewal of hematopoietic stem cells, which will reduce the efficacy, that is, it may not achieve the purpose of life-long cure.
  • the high-concentration, high-quality lentivirus required for clinical use requires extremely high equipment and technology, so it is difficult to reduce the cost. Therefore, a parallel, safer, and lower-cost clinical program is very necessary.
  • the ideal gene therapy method is to repair or destroy the traditional thalassemia mutation in the DNA of the patient’s hematopoietic stem cells to restore gene function, and permanently produce wild-type adult ⁇ -globin under the action of endogenous transcription control factors, thereby normal differentiation For 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 extremely low. Repairing point mutations requires high-efficiency HDR efficiency.
  • the present invention only needs to target the cutting target DNA to realize the NHEJ mutation and the strategy of destroying the mutation site is more feasible.
  • Clinically only need to transplant the autologous hematopoietic stem cells repaired by NHEJ method after gene editing back into the body to achieve the purpose of cure.
  • the present invention provides a method, product and application for repairing abnormal splicing of introns caused by mutations of ⁇ -globin gene (HBB) IVS2-654 C>T.
  • HBB ⁇ -globin gene
  • the present invention uses the CRISPR-Cas9 system to repair the abnormal splicing of introns caused by IVS2-654 C>T.
  • Cas9 targets the cleavage site
  • the 3 bases immediately adjacent to the 3'end of the target must be in the form of 5'-NGG-3' in the selection of the target sequence to form the PAM recognized by Cas9 itself. (protospacer adjacent motif) structure.
  • PAM protospacer adjacent motif
  • the inventor has conducted in-depth research on the abnormal splicing of introns caused by IVS2-654 C>T and found that the mutation of IVS2-654 C>T will cause an additional splice donor site in the second intron of HBB.”
  • "AAGGTAATA” which resulted in the production of an abnormal ⁇ -globin mRNA with an extra 73 bases, causing the translation to be terminated prematurely.
  • the destruction or deletion of the extra splice donor site caused by IVS2-654 C>T in the HBB gene region so that it will not cause abnormal variable splicing, so that this genomic region can be transcribed to produce normal ⁇ -beads Protein mRNA, and translated to produce normal ⁇ -globin.
  • the additional splice donor site "AAGGTAATA" caused by IVS2-654 C>T can be destroyed by base insertion, deletion, change, frameshift mutation or knockout.
  • the CRISPR-Cas9 system can be used to destroy the additional splice donor site "AAGGTAATA" caused by IVS2-654 C>T; preferably, when the CRISPR-Cas9 system is used, the designed sgRNA targeting sequence Preferably, it is within the range of 20 bp upstream of the IVS2-654 C>T site to 70 bp downstream of the IVS2-654 C>T site.
  • the CRISPR-Cas9 system can be used to simultaneously target the upstream and downstream ends or near both ends of the entire intron, accompanied by base insertion , Deletion, alteration, frameshift mutation or knockout methods cause large fragments of the gene region containing the disease-causing site to be deleted in the intron region, which can directly and efficiently destroy or delete the additional splicing supply caused by IVS2-654 C>T Body position "AAGGTAATA".
  • 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 In comparison, the system is simpler and more convenient to operate.
  • CRISPR 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 In comparison, the system is simpler and more convenient to operate.
  • RNA-guided endonucleases RNA-guided endonucleases
  • RGENs RNA-guided endonucleases
  • the former is the fusion of CRISPR RNAs (crRNAs) and trans-activating crRNA (tracrRNA) in the naturally occurring type II CRISPR-Cas system into single-stranded guide RNA (sgRNA) ,thereby:
  • DSB double strand break
  • the Cas9 may be selected from Cas9 from Streptococcus pyogenes, Staphylococcus aureus or N. meningitidis.
  • the Cas9 may 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.
  • the IVS2-654 C>T site upstream 20bp to the IVS2-654 C>T site Cleavage is performed in the range of 70 bp downstream. This cleavage will cause the extra translational abnormality of the splice donor site "AAGGTAATA" generated by IVS2-654 C>T to achieve the repair effect.
  • the present invention after Cas9 cuts the target sequence, there is no need to introduce foreign DNA donor sequences for homologous recombination repair.
  • sgRNA-1 is used to cut the target sequence, and the target sequence of sgRNA-1 is CAGTGATAATTTCTGGGTTA (SEQ ID No. 1), and sgRNA-1 can guide Cas9 at the IVS2-654 C>T site
  • the DNA is cut at 6 bases upstream, and the separate cutting results in the loss of bases, so that the additional splice donor site caused by IVS2-654 C>T causes the loss of additional bases and destroys the additional splice donor site.
  • sgRNA-1 can also be used in combination with sgRNA-2.
  • the targeting sequence of sgRNA-2 is TAAATTGTAACTGATGTAAG (SEQ ID No. 2), and sgRNA-2 can guide Cas9 in IVS2-654. DNA is cut at 53 bases downstream of the T site; the combination of sgRNA-1 and sgRNA-2 can also destroy the extra splice donor site caused by IVS2-654 C>T.
  • sgRNA-1 is used alone or sgRNA-1 and sgRNA-2 are used at the same time, the function of the additional splicing donor site can be disrupted, so that it will not cause abnormal alternative splicing, so that this genomic region can be transcribed Normal ⁇ -globin mRNA, and translated into normal ⁇ -globin.
  • sgRNA1 or sgRNA1+2 targeted gene editing is performed on the second intron region of normal human HBB, and the corresponding base loss or large fragment DNA deletion occurs, it will not affect the normal shearing or transcription of ⁇ -globin mRNA . This shows that when editing the disease-causing allele, even if another normal allele is edited at the same time, the expression of ⁇ -globin will not be affected.
  • the CRISPR-Cas9 system is used to target the upstream and downstream ends respectively, that is, the entire intron range for cutting, accompanied by base insertion, deletion, change, Frame-shift mutations or knockouts cause large fragment deletions in the gene region containing the pathogenic site IVS2-654 C>T in the intron region, which can directly and efficiently destroy or delete the additional splicing supply caused by IVS2-654 C>T Body position "AAGGTAATA", so as to achieve the effect of repair.
  • Cas9 cuts the target sequence, there is no need to introduce foreign DNA donor sequences for homologous recombination repair.
  • a combination of sgRNA-U and sgRNA-D is used to cut the target sequence, and the target sequence of sgRNA-U is 5'-TTCTTTCCCCTTCTTTTCTA-3' (SEQ ID No. 3), sgRNA-D The targeting sequence of 5'-GATTATTCTGAGTCCAAGCT-3' (SEQ ID No.
  • sgRNA-U and sgRNA-D can guide Cas9 to cut DNA at the upstream and downstream of the pathogenic site IVS2-654 C>T simultaneously, resulting in the inclusion of The large fragment bases within the pathogenic site are lost, so that the extra splice donor site caused by IVS2-654 C>T is efficiently destroyed and deleted, and the function of the extra splice donor site is lost.
  • the present invention provides a method for repairing abnormal splicing of introns caused by the mutation of HBB ( ⁇ -globin gene) IVS2-654 C>T in cells.
  • the IVS2-654 C>T can cause additional
  • the method comprises the step of gene-editing HBB using the CRISPR-Cas9 system to inactivate or delete the additional splice donor site;
  • the CRISPR-Cas9 system includes Cas9 and at least one sgRNA targeting a target sequence.
  • the inactivation includes the destruction of the function of the above-mentioned additional splice donor site through base insertion, deletion, change, frameshift mutation or knockout.
  • the target sequence of the sgRNA is 20 bp upstream of the IVS2-654 C>T site to 70 bp downstream of the IVS2-654 C>T site.
  • the sgRNA includes sgRNA-1; in other embodiments, the sgRNA includes sgRNA-1 and sgRNA-2.
  • the targeting sequence of the sgRNA-1 is CAGTGATAATTTCTGGGTTA (SEQ ID No. 1); the targeting sequence of the sgRNA-2 is TAAATTGTAACTGATGTAAG (SEQ ID No. 2).
  • the sgRNA includes sgRNA-U and sgRNA-D,
  • the targeting site of the sgRNA-U is within the range of 21bp upstream of the IVS2-654 C>T site to the start site of the second intron of the HBB gene where the IVS2-654 C>T site is located,
  • the targeting site of the sgRNA-D is within the range of 71 bp downstream of the IVS2-654 C>T site to the termination site of the second intron of the HBB gene where the IVS2-654 C>T site is located.
  • the targeting sequence of the sgRNA-U is any one or two or more of the sequences shown in SEQ ID No. 3-37; preferably, the sequence shown in SEQ ID No. 3, 4, and/or 5 Show sequence.
  • the sgRNA-D targeting sequence is any one or two or more of the sequences shown in SEQ ID Nos. 38-53; preferably, SEQ ID Nos. 38, 39, 40 and/or 41 Sequence shown.
  • sequence of the additional splice donor site is AAGGTATAATA.
  • the electrotransformation method is used to introduce a complex containing Cas9 and sgRNA into the cell.
  • the cells are hematopoietic stem cells or erythroid progenitor cells,
  • the hematopoietic stem cells are CD34 + hematopoietic stem progenitor cells, and the erythroid progenitor cells are HUDEP-2,
  • the cell is an isolated cell.
  • 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 few bases; the n is selected from 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • 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.
  • the first base, the second base, the third base, the fourth base, the fifth base, the 1-2 base, the 1-3 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, 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.
  • an electrotransformation method is used to introduce a complex containing Cas9 and sgRNA into the cell.
  • the molar ratio of Cas9 and sgRNA is 1:1-3, preferably, 1:2, more preferably 1:3.
  • 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 each stage of differentiation, RNA is extracted, reverse transcription is cDNA gel electrophoresis to detect abnormal splicing of introns, and to verify whether the repaired CDS region of hematopoietic stem cells can restore normal coding function.
  • the present invention also provides a sgRNA for repairing abnormal splicing of introns caused by HBB ( ⁇ -globin gene) IVS2-654 C>T mutation,
  • the sgRNA includes sgRNA-1, and the targeting sequence of the sgRNA-1 is CAGTGATAATTTCTGGGTTA (SEQ ID No. 1);
  • the sgRNA also includes sgRNA-2, and the targeting sequence of the sgRNA-2 is TAAATTGTAACTGATGTAAG (SEQ ID No. 2); or,
  • the sgRNA includes sgRNA-U and sgRNA-D,
  • the targeting site of the sgRNA-U is within the range of 21 bp upstream of the IVS2-654 C>T site to the start site of the second intron of the HBB gene where the IVS2-654 C>T site is located;
  • the targeting site of the sgRNA-D is within the range of 71 bp downstream of the IVS2-654 C>T site to the termination site of the second intron of the HBB gene where the IVS2-654 C>T site is located.
  • the targeting sequence of the sgRNA-U is any one or two or more of the sequences shown in SEQ ID No. 3-37; preferably, the sequence shown in SEQ ID No. 3, 4, and/or 5 Show the sequence, more preferably, the sgRNA-U includes chemical modification of base;
  • the sgRNA-D targeting sequence is any one or two or more of the sequences shown in SEQ ID No. 38-53; preferably, the sequences shown in SEQ ID No. 38, 39, 40 and/or 41 ; More preferably, the sgRNA-D includes base chemical modification.
  • the present invention also provides the application of the above-mentioned sgRNA in repairing abnormal splicing of introns caused by HBB ( ⁇ -globin gene) IVS2-654 C>T mutation.
  • the cells are hematopoietic stem cells or erythroid progenitor cells, preferably, the hematopoietic stem cells are CD34 + hematopoietic stem progenitor cells, and the erythroid progenitor cells are HUDEP-2; more preferably, the cells are Isolated cells.
  • the present invention designs sgRNA for the non-targeted region of IVS2-654 C>T, and also designs sgRNA for the second intron region of the HBB gene where IVS2-654 C>T is located.
  • the present invention is designed for more precise genomics And flexible editing provides the possibility, the mutation efficiency can be as high as 95%, which is significantly higher than the mutation rate that can be achieved by using ZFN, TALEN or Cas12a/Cpf1 RNP.
  • the high mutation efficiency achieved by the present invention has great significance in terms of saving experimental time and manpower and material resources.
  • the present invention uses a method for rapidly constructing mutations near the pathogenic site of hematopoietic stem cells in patients with ⁇ -thalassemia IVS2-654 C>T, or upstream and downstream to the two ends of the second intron, and directly introduces cleavable stem cells into defective hematopoietic stem cells. Or delete the guide RNA and CAS9 protein of the pathogenic site, and the damage and deletion of the pathogenic site can be quickly and efficiently caused by DNA double-strand break (DSB).
  • the ⁇ -globin intron obtained by the invention can be spliced normally, and the CDS region recovers its coding function.
  • Figure 1 is a schematic diagram of the principle of the CRISPR/Cas9 system.
  • Figure 2 is a schematic diagram of the abnormal splicing mutation site of ⁇ -thalassemia IVS2-654 C>T.
  • Figure 3 is a schematic diagram of the patient's hematopoietic stem cell pathogenic site editing, extracting cell genomic DNA and PCR amplifying the corresponding genomic region for Sanger sequencing.
  • the above figure is a blank control group (IVS2-654 patient-derived hematopoietic stem cells, without any RNP ), the sequencing peak diagram is normal; the middle diagram is the electrotransformation sgRNA-1 group, the sequencing peak diagram shows that the random number of bases is lost from the sgRNA-1 cleavage site and the spurious peaks are generated; the bottom diagram is electrotransmission sgRNA-1+sgRNA- In the 2 groups, the sequencing peak map showed that the random number of bases was lost from the sgRNA-1 cleavage site and the noise peak was generated.
  • Figure 4 is a gel electrophoresis diagram of cDNA PCR detection of the patient's hematopoietic stem cell pathogenic site editing and red blood cell differentiation.
  • the band with slower migration on the upper side (468bp) is the amplified product of abnormal shearing
  • the band with slower migration on the lower side (395bp) is the amplified product of normal shearing. It can be seen from the figure that the edited sample is abnormally sheared The ratio of the strength of the normal shear band is significantly reduced.
  • Lanes 1 and 6 are DNA molecular size standards
  • lanes 2-5 are samples of red blood cell differentiation to 11 days in vitro (cultured in EDM-2 medium)
  • lanes 7-10 are samples of red blood cell differentiation to 18 days in vitro (cultured in EDM -3 medium).
  • HU-2 represents in vitro differentiated erythrocytes derived from healthy people
  • IVS2-654 represents in vitro differentiated erythrocytes derived from patients with thalassaemia
  • 654-sg1 or 654-sg1+2 represents Cas9-edited in vitro differentiated erythrocytes.
  • Figure 5 shows the patient's hematopoietic stem cell pathogenic site editing and red blood cell differentiation after PCR amplification of HBB sequence from complete cDNA and Sanger sequencing; the above figure is a blank control group (IVS2-654 patient-derived hematopoietic stem cells, without any RNP) ,
  • the sequencing peak map shows that at the splicing of the second and third exons, a double peak is generated due to an abnormal splicing of an allele; the middle image is the electrotransformed sgRNA-1 group.
  • the sequencing peak image shows that the main peak is the normal HBB sequence, with only extreme Double peaks caused by low shear abnormalities; the figure below is the electrotransformed sgRNA-1+sgRNA-2 group.
  • the sequencing peak diagram shows that the main peak is the normal HBB sequence, and only the double peaks caused by extremely low shear abnormalities.
  • Fig. 6 is a schematic diagram of globin qPCR after the pathogenic site mutation and differentiation of patient hematopoietic stem cells.
  • the results showed that the mRNA ratio of HBB and HBA for cells edited by sgRNA-1 was about 50%, while for cells edited by sgRNA-1+2, the mRNA ratio of HBB and HBA was close to 100%. This ratio is sufficient to eliminate red blood cell toxicity caused by excessive HBA levels and can effectively alleviate the symptoms of thalassemia.
  • Figure 7 is a gel electrophoresis diagram of the target sequence in the genomic DNA of the patient's hematopoietic stem cells amplified by PCR. Among them, from left to right, lane 1 is the DNA molecular size standard, lane 2 is the amplified fragment of the blank control (1388bp); lane 3 is the amplified fragment of electrotransformed sg-U+D.
  • Fig. 8 is a gel electrophoresis diagram of the HBB exon in the hematopoietic stem cell cDNA amplified by PCR.
  • lane 1 is the DNA molecular size standard
  • lanes 2-4 are samples of red blood cells differentiated to 18 days in vitro.
  • 654 represents the in vitro differentiated erythrocytes derived from patients with thalassaemia
  • sg-U+D represents the in vitro differentiated erythrocytes after Cas9 editing
  • CD34 + represents the in vitro differentiated erythrocytes derived from healthy people.
  • the upper side of the band A (468bp) with slower migration is abnormal
  • the amplified product of splicing, the band N (395bp) which migrates faster on the lower side is the amplified product of normal splicing.
  • Figure 9 shows the sequencing results of the CDS region of the patient's hematopoietic stem cells HBB amplified by PCR.
  • the upper picture is the result of the blank control
  • the sequencing peak picture shows that the splicing of the second and third exons caused a double peak due to an abnormal splicing of an allele
  • the middle picture is the result of electrotransformation of sg-U+D
  • sequencing The peak figure shows that the main peak is the normal HBB sequence, and there are basically no double peaks caused by abnormal shearing; the figure below shows hematopoietic stem cells from healthy people.
  • Fig. 10 is a schematic diagram of globin qPCR after the pathogenic site mutation and differentiation of patient hematopoietic stem cells.
  • 654 represents the result of in vitro differentiation of red blood cells derived from patients with thalassaemia
  • sg-U+D represents the result of in vitro differentiation of red blood cells after Cas9 editing.
  • Figure 11 is a gel electrophoresis image of PCR amplification of target sequences in genomic DNA in HUDEP-2 cells. Among them, from left to right, lane 1 is the blank control result; lanes 2-5 are the results of treatments 1-4, respectively.
  • Figure 12 is a gel electrophoresis diagram of the HBB exon in the HUDEP-2 cell cDNA amplified by PCR. Among them, from left to right, lane 1 is the DNA molecular size standard, and HUDEP-2 in lane 2 represents the result of the blank control; lanes 3-6 deal with the results of 1-4 respectively.
  • the present invention uses CRISPR-Cas9 gene editing technology to target and destroy the abnormal mutation site IVS2-654 C>T in ⁇ -thalassemia (as shown in Figure 2), and construct a guide RNA that can identify and guide the Cas9 protein to the target sequence of the target gene 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, and simultaneously identifying the target genomic DNA sequence And cutting (as shown in Figure 1).
  • the cells are cultured in vitro to express the nuclease and cause double-strand breaks in the target genomic DNA near or at both ends of the pathogenic site (in the second intron gene region), resulting in including the pathogenic site Delete the large fragment.
  • the pathogenic site in the second intron gene region
  • the construction method of the present invention for the destruction of pathogenic sites in IVS2-654 C>T-deficient hematopoietic stem cells includes the following steps:
  • the EDM-2 stage differentiation is performed for 4 days, and the EMD-3 stage differentiation is performed for 7 days.
  • RNA is extracted, reverse transcribed into cDNA, gel electrophoresis to detect abnormal splicing of introns, and qPCR to detect the content and ratio of edited globin mRNA, and verify whether the repaired hematopoietic stem cell CDS region can restore normal coding function .
  • hematopoietic stem cells refer to hematopoietic stem progenitor cells (CD34 + HSPCs) of patients with ⁇ -thalassemia IVS2-654 C>T genotype. Electrotransform the above-mentioned chemically modified sgRNA and Cas9 protein mixture to ⁇ -thalassemia IVS2-654 C>T hematopoietic stem cells to efficiently destroy or delete abnormal splicing mutation sites, thereby restoring ⁇ -globin gene expression.
  • the present invention can use the existing gene editing technology to repair the transfusion-dependent ⁇ -thalassemia IVS2-654 C>T, has high editing efficiency, and can efficiently modify the autologous hematopoietic stem cells to permanently balance the hematopoietic system.
  • the patient is a ⁇ -thalassemia double heterozygous, and the genotype is IVS2-654/CD17.
  • This example is based on the IVS2-654 pathogenic site without suitable PAM targeted cleavage and destruction of abnormal splicing sites, so it is Design sgRNA-1 and sgRNA-2 before and after the disease site:
  • sgRNA-1 targeting sequence CAGTGATAATTTCTGGGTTA (SEQ ID No. 1);
  • sgRNA-2 targeting sequence TAAATTGTAACTGATGTAAG SEQ ID No. 2;
  • sgRNA-1 alone can cause breakage repair before the disease-causing mutation site, and sgRNA-1 and sgRNA-2 can work together to produce large fragment deletions before and after the disease-causing site.
  • the sgRNA and Cas9 protein synthesized by chemical modification were mixed in a certain proportion and incubated at room temperature for 10 minutes, and electrotransformed sgRNA-1 (654-sg1), sgRNA-1+sgRNA-2 (654-sg1+2), blank control (not added) after any RNP cells), by electroporation kit proportions electroporation liquid quantity electroporation cells does not exceed 10 5, the cells were centrifuged electroporation resuspended cells with the above-described incubated sgRNA and Cas9 protein complex mix gently transferred to The electroporation cup, to avoid air bubbles during the operation, use the CD34 cell electroporation program EO-100 for electroporation (Lonza-4D electroporation instrument).
  • the mutation rate of the target site after electroporation can be as high as 95%.
  • 654-check-F primer sequence CACATATTGACCAAATCAGGG (SEQ ID No. 54); 654-check-R primer sequence: CTTTGCCAAAGTGATGGGCCA (SEQ ID No. 55).
  • the differentiation can be continued in the EDM-2 medium.
  • the cells can be expanded in large quantities.
  • the differentiation stage of EDM-2 is over, the appropriate amount of cells are collected, RNA is extracted and reverse transcribed into cDNA, and the remaining cells are continued to differentiate into EDM-3. After the differentiation, the RNA is extracted and reversed into cDNA.
  • the sgRNA-1 edited cells, HBB and HBA mRNA ratio is about 50%, and sgRNA-1+2 In edited cells, the mRNA ratio of HBB and HBA is close to 100%. This ratio is sufficient to eliminate red blood cell toxicity caused by excessive HBA levels and can effectively alleviate the symptoms of thalassemia.
  • primer sequences used are as follows:
  • 654-exon1-F primer sequence TGAGGAGAAGTCTGCCGTTAC (SEQ ID No. 56);
  • 654-exon3-R primer sequence CACCAGCCACCACTTTCTGA (SEQ ID No. 57);
  • HBB-CDS-F primer sequence ATGGTGCATCTGACTCCTGA (SEQ ID No. 58);
  • HBB-CDS-R primer sequence TTAGTGATACTTGTGGGCCA (SEQ ID No.59);
  • HBA-S_qPCR primer sequence GCCCTGGAGAGGATGTTC (SEQ ID No. 60);
  • HBA-A_qPCR primer sequence TTCTTGCCGTGGCCCTTA (SEQ ID No. 61);
  • HBB-S_qPCR primer sequence TGAGGAGAAGTCTGCCGTTAC (SEQ ID No. 62);
  • HBB-AS_qPCR primer sequence ACCACCAGCAGCCTGCCCA (SEQ ID No. 63);
  • HBG-S_qPCR primer sequence GGTTATCAATAAGCTCCTAGTCC (SEQ ID No. 64);
  • HBG-AS_qPCR primer sequence ACAACCAGGAGCCTTCCCA (SEQ ID No. 65);
  • HBB_e2-e3 primer sequence TTCAGGCTCCTGGGCAAC (SEQ ID No.66);
  • R_HBB_exon3 primer sequence CACCAGCCACCACTTTCTGA (SEQ ID No. 67).
  • the ⁇ -thalassemia IVS2-654 C>T-deficient hematopoietic stem cells used in this example were derived from a patient with a double heterozygous genotype of IVS2-654 C>T with a CD17 mutation.
  • the sgRNA was designed based on the sequence from 21bp upstream of the IVS2-654 C>T pathogenic site to the start site of the second intron of the HBB gene where the IVS2-654 C>T site is located, named sgRNA-U, and numbered sgRNA-U1, 2, 3,...;
  • sgRNA based on the sequence from 71bp downstream of the IVS2-654 C>T pathogenic site to the termination site of the second intron of the HBB gene where the IVS2-654 C>T site is located, named sgRNA-D and numbered sgRNA -D1, 2, 3,...
  • One sgRNA-U and one sgRNA-D work together to produce the deletion of corresponding fragments before and after the pathogenic site.
  • the above-mentioned targeting sequences of sgRNA-U and sgRNA-D are shown in Table 1.
  • Targeting sequence SEQ ID No. sgRNA-U1 TTCTTTCCCCTTCTTTTCTA 3 sgRNA-U2 CTTCTTTTCTATGGTTAAGT 4 sgRNA-U3 ATAATCATTATACATATTTA 5 sgRNA-U4 GAACTTAACCATAGAAAAGA 6 sgRNA-U5 ACTTAACCATAGAAAAGAAG 7 sgRNA-U6 TATGGTTAAGTTCATGTCAT 8 sgRNA-U7 GTTAAGTTCATGTCATAGGA 9 sgRNA-U8 TTAAGTTCATGTCATAGGAA 10 sgRNA-U9 TAAGTTCATGTCATAGGAAG 11 sgRNA-U10 ATAGGAAGGGGATAAGTAAC 12
  • sgRNA-U11 TAGGAAGGGGATAAGTAACA 13
  • sgRNA-U12 TAACAGGGTACAGTTTAGAA 14
  • sgRNA-U13 AACAGGGTACAGTTTAGAAT 15
  • sgRNA-U14 GACGAATGATTGCATCAGTG 16
  • sgRNA-U15 TGCATCAGTGTGGAAGTCTC 17
  • sgRNA-U16 AAGTATAATAGTAAAAATTG
  • sgRNA-U17 TTTTGTTATACACAATGTTA 19
  • sgRNA-U18 TTAACATTGTGTATAACAAA 20
  • sgRNA-D13 GTATGAACATGATTAGCAAA 50
  • sgRNA-D14 ACCATAAAATAAAAGCAGAA 51
  • sgRNA-D15 CGTTGCCCAGGAGCTGTGGG 52
  • sgRNA-D16 GCACGTTGCCCAGGAGCTGT 53
  • a total of 51 sgRNA-U and sgRNA-D in Table 1 were synthesized by chemical modification.
  • sgRNA-U or sgRNA-D synthesized by chemical modification and Cas9 protein are mixed in a certain proportion and incubated at room temperature for 10 minutes.
  • different combinations of sgRNA-U and sgRNA-D (hereinafter referred to as sg-U+D) are transferred to ⁇ -ground.
  • sgRNA-U1 (or sgRNA-U16 or sgRNA-U32) and sgRNA-D1
  • sgRNA-U2 (or sgRNA-U17 or sgRNA-U33) and sgRNA-D2
  • sgRNA-U2 (or sgRNA-U18 or sgRNA-U34) and sgRNA-D3,
  • sgRNA-U3 (or sgRNA-U19 or sgRNA-U35) and sgRNA-D4,
  • sgRNA-U4 (or sgRNA-U20) and sgRNA-D5
  • sgRNA-U4 (or sgRNA-U20) and sgRNA-D5
  • sgRNA-U5 (or sgRNA-U21) and sgRNA-D6,
  • sgRNA-U6 (or sgRNA-U22) and sgRNA-D7,
  • sgRNA-U7 (or sgRNA-U23) and sgRNA-D8,
  • sgRNA-U8 (or sgRNA-U24) and sgRNA-D9
  • sgRNA-U9 (or sgRNA-U25) and sgRNA-D10
  • sgRNA-U10 (or sgRNA-U26) and sgRNA-D11
  • sgRNA-U11 (or sgRNA-U27) and sgRNA-D12,
  • sgRNA-U12 (or sgRNA-U28) and sgRNA-D13,
  • sgRNA-U13 or sgRNA-U29
  • sgRNA-D14 sgRNA-D14
  • sgRNA-U14 (or sgRNA-U30) and sgRNA-D15,
  • sgRNA-U15 (or sgRNA-U31) and sgRNA-D16.
  • step 3 of step 1 After the hematopoietic stem cells obtained in step 3 of step 1 were differentiated and cultured in vitro for 3-4 days, an appropriate amount of cells were collected, the genome was extracted, and the amplified products were detected by gel electrophoresis after PCR amplification to identify mutation efficiency and DNA fragment deletion status. The remaining cells continued to differentiate in EDM-1 medium until the 7th day.
  • primer sequences used in PCR are as follows:
  • 654-U+D-F 5’-GCTTCTGACACAACTGTGTTC-3’ (SEQ ID No. 68);
  • 654-U+D-R 5'-CTTTGCCAAAGTGATGGGCCA-3' (SEQ ID No. 69).
  • the differentiation can be continued in EDM-2 medium.
  • the cells can be expanded in large quantities, and some cells can be frozen in advance for later use.
  • the remaining cells are transferred to EDM-3 to continue differentiation.
  • the RNA is extracted and reversed into cDNA for next use. The detection in steps 3-5 is described.
  • Method Use primers 654-exon1-F and 654-exon3-R to amplify differentiated exons by PCR to verify whether the disease-causing site can be spliced normally after mutation.
  • the HBB coding region sequence was PCR amplified from the complete cDNA and Sanger sequenced; the primers used were HBB-CDS-F: and HBB-CDS-R.
  • Methods qPCR analysis of changes in globin content in hematopoietic stem cells after pathogenic site mutations.
  • the primers used in qPCR are: HBA-S, HBA-A, HBB_e2-e3, R_HBB_exon3, HBG-S and HBG-AS.
  • results In patients with hematopoietic stem cells edited by sgRNA-U and sgRNA-D, the mRNA ratio of HBB to HBA is close to 60%, which is sufficient to eliminate the red blood cell toxicity caused by excessive HBA levels and can effectively relieve the symptoms of thalassemia.
  • electrotransformation sg The results of -U+D (specifically sgRNA-U1 and sgRNA-D1) are shown in FIG. 10.
  • the cells used in this example are HUDEP-2 cells (Human Umbilical Cord Blood Derived Erythoid Progenitor-2, erythroid progenitor cells derived from human umbilical cord blood).
  • Treatment 4 HUDEP-2 cells electrotransfected with sgRNA-U3 and sgRNA-D4.

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Abstract

提供一种内含子异常剪接的修复方法、产品和应用。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)中异常突变位点IVS2-654 C>T，通过设计并合成能识别引导Cas9蛋白至目标基因靶序列的引导RNA序列(sgRNA)，将其与Cas9蛋白混合物电转导入β-地贫IVS2-654 C>T造血干细胞中，高效破坏或删除该异常剪接突变位点，从而恢复β-珠蛋白基因的正常剪切和表达。该方法编辑效率高，能高效修饰自体造血干细胞，持久平衡造血***。

Description

一种内含子异常剪接的修复方法、产品和应用 技术领域
本发明涉及基因工程技术领域，具体涉及一种内含子异常剪接的修复方法、产品和应用。
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***由Clustered regularly interspaced short palindromic repeats(CRISPR)和CRISPR-associated(CAS)基因组成。CRISPR***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***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。
Clustered regularly interspaced short palindromic repeats(CRISPR)***分为I，II，III型三个家族，其中II型***仅需要Cas9蛋白即可在反式编码小RNA(trans-encoded small RNA，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)，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***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，IVS2-654 C>T基因型是我国“地贫”中较为多见的一种，发病机制为HBB基因第2内含子中的第654位碱基发生C>T突变，从而产生异常剪接位点，导致β-珠蛋白mRNA中额外多出了73nt的外显子并提前终止翻译。
目前，中间型和重型地贫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原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病人造血干细胞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)所占的比例极低，修复点突变需要高效的HDR效率。
本发明针对致病位点的异常突变，仅需靶向切割目标DNA实现NHEJ突变即可破坏突变位点的策略显得更具可行性。在临床上，只需将基因编辑后通过NHEJ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。
发明内容
本发明提供了一种针对β-珠蛋白基因(HBB)IVS2-654 C>T的突变导致的内含子异常剪接的修复方法、产品及应用。
本发明利用CRISPR-Cas9***对IVS2-654 C>T导致的内含子异常剪接进行修复。Cas9靶向切割位点时，需要在可供靶向序列的选择上与靶点3′端紧邻的3个碱基必须是5′-NGG-3′的形式，从而构成被Cas9本身识别的PAM(protospacer adjacent motif)结构。但是，上述致病突变位点附近并没有合适的PAM靶向切割破坏异常剪接位点，这导致无法利用CRISPR-Cas9***直接切割IVS2-654 C>T突变位点并进行修复。
发明人针对IVS2-654 C>T导致的内含子异常剪接进行了深入的研究，发现IVS2-654 C>T的突变会使得HBB第二个内含子中产生一个额外的剪接供***点“AAGGTAATA”，导致产生一个多出73个碱基的异常的β-珠蛋白mRNA，使得翻译被提前终止。理论上，在HBB基因区域破坏或删除IVS2-654 C>T导致的额外的剪接供***点，使得其不会引发异常可变剪切，就可以使得此基因组区域可以转录产生正常的β-珠蛋白mRNA，并翻译产生正常的β-珠蛋白。
在一个实施方式中，可以采用碱基***、缺失、改变、移码突变或敲除的方式破坏由于IVS2-654 C>T导致的额外的剪接供***点“AAGGTAATA”。
在一个实施方式中，可以采用CRISPR-Cas9***破坏由于IVS2-654 C>T导致的额外的剪接供***点“AAGGTAATA”；优选的，采用CRISPR-Cas9***时，所设计的sgRNA的靶向序列优选在IVS2-654 C>T位点的上游20bp至IVS2-654 C>T位点的下游70bp范围内。
在另一个实施方式中，以致病位点IVS2-654 C>T为界，可以采用CRISPR-Cas9***分别同时靶向切割整个内含子上游和下游两端或两端附近，伴随碱基***、缺失、改变、移码突变或敲除的方式造成内含子区域包含致病位点的基因区大片段缺失，即可直接高效破坏或删除由于IVS2-654 C>T导致的额外的剪接供***点“AAGGTAATA”。
本发明中，所述CRISPR-Cas***是指适合被人工改造的CRISPR-Cas***、源于古细菌II型(CRISPR)-CRISPR-associated protein(Cas)***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***中的CRISPR RNAs(crRNAs)与trans-activating crRNA(tracrRNA)融合成单链引导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)进行修复可高效造成目标基因的移码，从而实现对致病突变位点的破坏；或与Cas9蛋白结合并引导后者对致病位点上游、下游两条目标DNA序列同时进行特异性的切割，切割将形成双链断裂(double strand break,DSB)，该损伤可造成包括致病位点在内的DNA大片段缺失，从而实现对致病突变位点的破坏或删除。
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。
本发明中，由于IVS2-654 C>T位点附近没有适合Cas9蛋白切割的靶向序列，因此，选择了在IVS2-654 C>T位点的上游20bp至IVS2-654 C>T位点的下游70bp范围内进行切割，此切割会引起由IVS2-654 C>T产生的额外的剪接供***点“AAGGTAATA”的翻译异常，从而达到修复的效果。本发明中，Cas9切割目标序列后，不需要引入外源的DNA供体序列进行同源重组修复。
在一个实施方式中，采用sgRNA-1进行目标序列的切割，所述sgRNA-1的靶向序列为CAGTGATAATTTCTGGGTTA(SEQ ID No.1)，sgRNA-1可以引导Cas9在IVS2-654 C>T位点上游6个碱基处切割DNA，单独切割导致碱基丢失，从而使得由于IVS2-654 C>T引起的额外的剪接供***点造成额外碱基丢失，破坏该额外的剪接供***点。
在另一个实施方式中，sgRNA-1还可以和sgRNA-2联用，所述sgRNA-2的靶向序列为TAAATTGTAACTGATGTAAG(SEQ ID No.2)，sgRNA-2可以引导Cas9在IVS2-654 C>T位点下游53个碱基处切割DNA；sgRNA-1和sgRNA-2联用同样可以破坏由于IVS2-654 C>T引起的额外的剪接供***点。
不管是单独使用sgRNA-1，还是同时使用sgRNA-1和sgRNA-2，都可以破坏额外的剪接供***点的功能，使得其不会引发异常可变剪切，从而使得此基因组区域可以转录产生正常的β-globin mRNA，并翻译产生正常的β-globin。
如果对正常人HBB第二内含子区域进行sgRNA1或者sgRNA1+2靶向的基因编辑，并产生相应的碱基丢失或者大片段DNA缺失，并不会影响β-globin  mRNA的正常剪切或转录。这说明在编辑导致疾病的等位基因时，即使同时编辑了另一条正常的等位基因，也不会影响β-globin的表达。
本发明中，以致病位点IVS2-654 C>T为界，采用CRISPR-Cas9***分别靶向上游、下游两端即整个内含子范围内进行切割，伴随碱基***、缺失、改变、移码突变或敲除的方式造成内含子区域包含致病位点IVS2-654 C>T的基因区大片段缺失，可直接高效破坏或删除由于IVS2-654 C>T导致的额外的剪接供***点“AAGGTAATA”，从而达到修复的效果。本发明中，Cas9切割目标序列后，不需要引入外源的DNA供体序列进行同源重组修复。
在一个实施方式中，采用sgRNA-U和sgRNA-D联用进行目标序列的切割，所述sgRNA-U的靶向序列为5’-TTCTTTCCCCTTCTTTTCTA-3’(SEQ ID No.3)，sgRNA-D的靶向序列为5’-GATTATTCTGAGTCCAAGCT-3’(SEQ ID No.38)，sgRNA-U和sgRNA-D可以引导Cas9在致病位点IVS2-654 C>T上下游分别同时切割DNA，导致包含致病位点在内的大片段碱基丢失，从而使得由于IVS2-654 C>T引起的额外的剪接供***点高效率破坏和删除，使得该额外的剪接供***点功能丧失。
由于IVS2-654 C>T此类地贫的突变位点发生在内含子基因区，理论上只要将此部分致病的剪接供体破坏或删除，就可以破坏额外剪接供***点的功能，使得其不会引发异常可变剪切，从而使得此基因组区域可以转录产生正常的β-珠蛋白mRNA，并翻译产生正常的β-珠蛋白。
实验结果表明，正常人HBB第二内含子区域内，如果对以致病位点为界，分别靶向上游、下游两端范围进行sgRNA U+D的基因编辑，切割并产生包含致病位点在内的额外剪接供体大片段DNA的缺失，并不会影响β-珠蛋白mRNA的正常剪接或转录。这说明在编辑导致疾病的等位基因时，即使同时编辑了另一条正常的等位基因，也不会影响β-珠蛋白的表达。
发明详述：
一方面，本发明提供了一种针对细胞中HBB(β-珠蛋白基因)IVS2-654 C>T的突变导致的内含子异常剪接的修复方法，所述IVS2-654 C>T可以引起额外的剪接供***点，所述方法包括利用CRISPR-Cas9***对HBB进行基因编辑以失活或删除所述额外的剪接供***点的步骤；
所述CRISPR-Cas9***包括Cas9和至少一条靶向目标序列的sgRNA。
所述失活包括通过碱基***、缺失、改变、移码突变或敲除的方式破坏上述额外的剪接供***点的功能。
进一步的，所述sgRNA的靶向序列为IVS2-654 C>T位点的上游20bp至IVS2-654 C>T位点的下游70bp。
在一个实施方式中，所述sgRNA包括sgRNA-1；在其他的实施方式中，所述sgRNA包括sgRNA-1和sgRNA-2。
所述sgRNA-1的靶向序列为CAGTGATAATTTCTGGGTTA(SEQ ID No.1)；所述sgRNA-2的靶向序列为TAAATTGTAACTGATGTAAG(SEQ ID No.2)。
或者，所述sgRNA包括sgRNA-U和sgRNA-D，
所述sgRNA-U的靶向位点在IVS2-654 C>T位点的上游21bp至IVS2-654 C>T位点所在的HBB基因第二内含子起始位点范围内，
所述sgRNA-D的靶向位点在IVS2-654 C>T位点的下游71bp至IVS2-654 C>T位点所在的HBB基因第二内含子终止位点范围内。
进一步的，所述sgRNA-U的靶向序列为SEQ ID No.3-37所示序列中的任一种或任两种以上；优选的，SEQ ID No.3、4、和/或5所示序列。
进一步的，所述sgRNA-D的靶向序列为SEQ ID No.38-53所示序列中的任一种或任两种以上；优选的，SEQ ID No.38、39、40和/或41所示序列。
进一步的，所述额外的剪接供***点的序列为AAGGTAATA。
进一步的，采用电转化的方法向细胞中引入包含Cas9和sgRNA的复合物。
进一步的，所述细胞为造血干细胞或红系祖细胞，
优选的，所述造血干细胞为CD34 +的造血干祖细胞，所述红系祖细胞为HUDEP-2，
更优选的，所述细胞为离体细胞。
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。
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的复合物。
进一步的，所述Cas9和sgRNA的摩尔比为1:1-3，优选的，1:2，更优选1:3。
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，将电转后的细胞培养在CD34 +EDM-1分化体系中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。每阶段分化结束后提取RNA, 反转录为cDNA凝胶电泳检测内含子的异常剪接，并验证修复后的造血干细胞CDS区是否可以恢复正常编码功能。
另一方面，本发明还提供了一种用于修复由HBB(β-珠蛋白基因)IVS2-654 C>T突变导致的内含子异常剪接的sgRNA，
所述sgRNA包括sgRNA-1，所述sgRNA-1的靶向序列为CAGTGATAATTTCTGGGTTA(SEQ ID No.1)；
进一步的，所述sgRNA还包括sgRNA-2，所述sgRNA-2的靶向序列为TAAATTGTAACTGATGTAAG(SEQ ID No.2)；或，
所述sgRNA包括sgRNA-U和sgRNA-D，
所述sgRNA-U的靶向位点在IVS2-654 C>T位点的上游21bp至IVS2-654 C>T位点所在的HBB基因第二内含子起始位点范围内；
所述sgRNA-D的靶向位点在IVS2-654 C>T位点的下游71bp至IVS2-654 C>T位点所在的HBB基因第二内含子终止位点范围内。
进一步的，所述sgRNA-U的靶向序列为SEQ ID No.3-37所示序列中的任一种或任两种以上；优选的，SEQ ID No.3、4、和/或5所示序列，更优选的，所述sgRNA-U包括碱基的化学修饰；
所述sgRNA-D的靶向序列为SEQ ID No.38-53所示序列中的任一种或任两种以上；优选的，SEQ ID No.38、39、40和/或41所示序列；更优选的，所述sgRNA-D包括碱基的化学修饰。
另一方面，本发明还提供了上述sgRNA在修复细胞中由HBB(β-珠蛋白基因)IVS2-654 C>T突变导致的内含子异常剪接中的应用。
进一步的，所述细胞为造血干细胞或红系祖细胞，优选的，所述造血干细胞为CD34 +的造血干祖细胞，所述红系祖细胞为HUDEP-2；更优选的，所述细胞为离体细胞。
有益效果：
本发明针对IVS2-654 C>T的非靶向区域设计了sgRNA，还针对IVS2-654 C>T所在的HBB基因第二内含子区域设计了sgRNA，本发明为在基因组上进行更为精确和灵活的编辑提供了可能，突变效率可高达95％，显著高于采用ZFN、TALEN或Cas12a/Cpf1 RNP所能达到的突变率。本发明通过所实现的高突变效率，在节省实验时间以及人力物力的投入方面有很大的意义。
本发明通过在β-地贫IVS2-654 C>T患者造血干细胞致病位点附近、或上下游至第二内含子两端快速构建突变的方法，向缺陷的造血干细胞中直接引入能切割或删除致病位点的引导RNA与CAS9蛋白，通过DNA双链断裂(double strand break,DSB)能快速高效造成致病位点的破坏和删除。利用本发明获得的β-珠蛋白内含子可正常剪接，CDS区恢复编码功能。
附图说明
图1为CRISPR/Cas9***作用原理示意图。
图2为β-地贫IVS2-654 C>T异常剪接突变位点示意图。
图3为患者造血干细胞致病位点编辑后，提取细胞基因组DNA并PCR扩增对应的基因组区域进行Sanger测序示意图，上图是电转空白对照组(IVS2-654病人来源造血干细胞，未导入任何RNP)，测序峰图正常；中图是电转sgRNA-1组，测序峰图显示从sgRNA-1切割位点处由于随机数目的碱基丢失而产生杂峰；下图是电转sgRNA-1+sgRNA-2组，测序峰图显示从sgRNA-1切割位点处由于随机数目的碱基丢失而产生杂峰。
图4为患者造血干细胞致病位点编辑并红细胞分化后cDNA PCR检测凝胶电泳图。上侧迁移较慢的条带(468bp)为异常剪切的扩增产物，下侧迁移较慢的条带(395bp)为正常剪切的扩增产物，由图可见编辑后的样品异常剪切与正常剪切条带强度比值显著降低。泳道1和6为DNA分子大小标准，泳道2-5为红细胞体外分化至11天的样品(培养于EDM-2培养基)，泳道7-10为红细胞体外分化至18天的样品(培养于EDM-3培养基)。HU-2代表健康人来源的体外分化红细胞，IVS2-654代表地贫病人来源的体外分化红细胞，654-sg1或者654-sg1+2代表Cas9编辑后的体外分化红细胞。
图5为患者造血干细胞致病位点编辑并红细胞分化后从完整cDNA中PCR扩增HBB序列并进行Sanger测序；上图是电转空白对照组(IVS2-654病人来源造血干细胞，未导入任何RNP)，测序峰图显示在第二和第三外显子的拼接处由于一条等位基因拼接异常，产生双峰；中图是电转sgRNA-1组，测序峰图显示主峰为正常HBB序列，只有极低的剪切异常导致的双峰；下图是电转sgRNA-1+sgRNA-2组，测序峰图显示主峰为正常HBB序列，只有极低的剪切异常导致的双峰。
图6为患者造血干细胞致病位点突变分化后珠蛋白qPCR示意图。结果显示，经sgRNA-1编辑的细胞，HBB和HBA的mRNA比值约为50％，而经sgRNA-1+2编辑的细胞，HBB和HBA的mRNA比值接近100％。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。
图7为PCR扩增患者造血干细胞基因组DNA中目标序列的凝胶电泳图。其中，从左至右起，泳道1为DNA分子大小标准，泳道2是空白对照的扩增片段(1388bp)；泳道3是电转sg-U+D的扩增片段。
图8为PCR扩增患者造血干细胞cDNA中HBB外显子的凝胶电泳图。其中，从左至右起，泳道1为DNA分子大小标准，泳道2-4为红细胞体外分化至18天的样品。654代表地贫患者来源的体外分化红细胞，sg-U+D代表Cas9编辑后的体外分化红细胞，CD34 +代表健康人来源的体外分化红细胞，上侧迁移较慢的条带A(468bp)为异常剪接的扩增产物，下侧迁移较快的条带N(395bp)为正常剪接的扩增产物。
图9为PCR扩增患者造血干细胞HBB的CDS区的测序结果。其中，上图是空白对照结果，测序峰图显示在第二和第三外显子的拼接处由于一条等位基因拼接异常，产生双峰；中图是电转sg-U+D的结果，测序峰图显示主峰为正常HBB序列，且基本没有剪切异常导致的双峰；下图是健康人来源造血干细胞。
图10为患者造血干细胞致病位点突变分化后珠蛋白qPCR示意图。其中，654代表地贫患者来源的体外分化红细胞的结果，sg-U+D代表Cas9编辑后的体外分化红细胞的结果。
图11为PCR扩增HUDEP-2细胞中基因组DNA中目标序列的凝胶电泳图。其中，从左至右起，泳道1为空白对照结果；泳道2-5分别为处理1-4的结果。
图12为PCR扩增HUDEP-2细胞cDNA中HBB外显子的凝胶电泳图。其中，从左至右起，泳道1为DNA分子大小标准，泳道2的HUDEP-2代表空白对照结果；泳道3-6分别处理1-4的结果。
具体实施方式
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IVS2-654 C>T(如图2所示)，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，对目标基因组DNA序列同时进行识别和切割(如图1所示)。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或两端(在第二内含子基因区内)的目标基因组DNA发生双链断裂，造成包含致病位点在内的大片段删除。本实施方式中，不需要引入外源DNA序列。
本发明在IVS2-654 C>T缺陷型造血干细胞中对致病位点破坏的构建方法，包括如下步骤：
(1)sgRNA设计；
(2)sgRNA合成；
(3)将上述sgRNA和Cas9蛋白按摩尔比1-2：1混合并电转导入β-地贫IVS2-654 C>T患者造血干细胞；
(4)将电转后的细胞培养在CD34 +EDM-1分化体系中7天，提取上述步骤所得细胞的基因组DNA，PCR扩增确定突变效率；
(5)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，凝胶电泳检测内含子的异常剪接以及qPCR检测编辑后的珠蛋白mRNA含量及比例，并验证修复后的造血干细胞CDS区是否可以恢复正常编码功能。
本发明中，造血干细胞是指β-地贫IVS2-654 C>T基因型患者造血干祖细胞(CD34 +HSPCs)。将上述经化学修饰的sgRNA与Cas9蛋白混合物电转至β-地贫IVS2-654 C>T造血干细胞，高效破坏或删除异常剪接突变位点，从而恢复β-珠蛋白基因的表达。本发明可利用现有基因编辑技术修复依赖输血型β-地贫IVS2-654 C>T，编辑效率高，并能高效修饰自体造血干细胞持久平衡造血***。
实施例1 在β-地贫IVS2-654 C>T缺陷型造血干细胞中对致病位点的破坏
1、sgRNA的设计
本实施例中患者为β-地贫双重杂合子，基因型为IVS2-654/CD17，本实施例基于IVS2-654致病位点没有合适的PAM靶向切割破坏异常剪接位点，因此在致 病位点前后分别设计sgRNA-1和sgRNA-2：
sgRNA-1靶向序列CAGTGATAATTTCTGGGTTA(SEQ ID No.1)；
sgRNA-2靶向序列TAAATTGTAACTGATGTAAG(SEQ ID No.2)；
其中sgRNA-1单独作用可在致病突变位点前造成断裂修复，sgRNA-1和sgRNA-2共同作用可在致病位点前后产生大片段缺失。
2、sgRNA、Cas9蛋白的制备
3、sgRNA以及Cas9蛋白复合物的电转
将经由化学修饰合成的sgRNA及Cas9蛋白按一定比例混合后室温孵育10min，分别电转sgRNA-1(654-sg1)、sgRNA-1+sgRNA-2(654-sg1+2)、空白对照(未加入任何RNP的细胞)，按电转试剂盒比例混合电转液，电转细胞数量不超过10 5个，细胞离心后电转液重悬细胞并与上述孵育的sgRNA和Cas9蛋白复合物轻轻混匀后转移至电转杯，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，此部分即完成本发明在缺陷型造血干细胞致病突变位点的破坏。
以下用以鉴定是否成功实现本发明在目标基因突变构建方法。
(1)基因组DNA的突变鉴定
将步骤3得到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测序检测突变效率，剩余细胞继续在EDM-1培养基中分化至第7天。如图3所示，电转后目标位点的突变率可高达95％。其中，654-check-F引物序列：CACATATTGACCAAATCAGGG(SEQ ID No.54)；654-check-R引物序列：CTTTGCCAAAGTGATGGGCCA(SEQ ID No.55)。
(2)EDM-2、EDM-3分化培养
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。此阶段细胞可大量扩增，待EDM-2分化阶段结束，收集适量细胞，提取RNA反转录为cDNA，同时剩余细胞至EDM-3中继续分化，待分化结束后提取RNA反转成cDNA。
(3)外显子、CDS区扩增、q-PCR
扩增分化后的外显子，验证致病位点突变后是否能正常剪接。如图4所示，相比缺陷型细胞，致病位点突变后的造血干细胞内含子基本恢复正常剪接；对HBB CDS区进行扩增，如图5所示，测序结果表明，相比缺陷细胞，致病位点突变后的造血干细胞HBB基因恢复正常编码功能。q-PCR分析致病位点突变后的造血干细胞珠蛋白含量变化，如图6所示，经sgRNA-1编辑的细胞，HBB和HBA的mRNA比值约为50％，而经sgRNA-1+2编辑的细胞，HBB和HBA的mRNA比值接近100％。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。
其中，所用引物序列如下：
654-exon1-F引物序列：TGAGGAGAAGTCTGCCGTTAC(SEQ ID No.56)；
654-exon3-R引物序列：CACCAGCCACCACTTTCTGA(SEQ ID No.57)；
HBB-CDS-F引物序列：ATGGTGCATCTGACTCCTGA(SEQ ID No.58)；
HBB-CDS-R引物序列：TTAGTGATACTTGTGGGCCA(SEQ ID No.59)；
HBA-S_qPCR引物序列：GCCCTGGAGAGGATGTTC(SEQ ID No.60)；
HBA-A_qPCR引物序列：TTCTTGCCGTGGCCCTTA(SEQ ID No.61)；
HBB-S_qPCR引物序列：TGAGGAGAAGTCTGCCGTTAC(SEQ ID No.62)；
HBB-AS_qPCR引物序列：ACCACCAGCAGCCTGCCCA(SEQ ID No.63)；
HBG-S_qPCR引物序列：GGTTATCAATAAGCTCCTAGTCC(SEQ ID No.64)；
HBG-AS_qPCR引物序列：ACAACCAGGAGCCTTCCCA(SEQ ID No.65)；
HBB_e2-e3引物序列：TTCAGGCTCCTGGGCAAC(SEQ ID No.66)；
R_HBB_exon3引物序列：CACCAGCCACCACTTTCTGA(SEQ ID No.67)。
实施例2、对β-地贫IVS2-654 C>T缺陷型造血干细胞中致病位点的删除
本实施例中使用的β-地贫IVS2-654 C>T缺陷型造血干细胞来自基因型为IVS2-654 C>T复合CD17突变的双重杂合患者。
一、方法
1、sgRNA的设计
基于IVS2-654 C>T致病位点的上游21bp至IVS2-654 C>T位点所在的HBB基因第二内含子起始位点范围内序列设计sgRNA，命名为sgRNA-U，编号为sgRNA-U1、2、3、……；
基于IVS2-654 C>T致病位点的下游71bp至IVS2-654 C>T位点所在的HBB基因第二内含子终止位点范围内序列设计sgRNA，命名为sgRNA-D，编号为sgRNA-D1、2、3、……。
一条sgRNA-U和一条sgRNA-D共同作用可在致病位点前后产生相应片段的删除。上述sgRNA-U和sgRNA-D的靶向序列如表1所示。
表1
名称 靶向序列(5’-3’) SEQ ID No.
sgRNA-U1 TTCTTTCCCCTTCTTTTCTA 3
sgRNA-U2 CTTCTTTTCTATGGTTAAGT 4
sgRNA-U3 ATAATCATTATACATATTTA 5
sgRNA-U4 GAACTTAACCATAGAAAAGA 6
sgRNA-U5 ACTTAACCATAGAAAAGAAG 7
sgRNA-U6 TATGGTTAAGTTCATGTCAT 8
sgRNA-U7 GTTAAGTTCATGTCATAGGA 9
sgRNA-U8 TTAAGTTCATGTCATAGGAA 10
sgRNA-U9 TAAGTTCATGTCATAGGAAG 11
sgRNA-U10 ATAGGAAGGGGATAAGTAAC 12
sgRNA-U11 TAGGAAGGGGATAAGTAACA 13
sgRNA-U12 TAACAGGGTACAGTTTAGAA 14
sgRNA-U13 AACAGGGTACAGTTTAGAAT 15
sgRNA-U14 GACGAATGATTGCATCAGTG 16
sgRNA-U15 TGCATCAGTGTGGAAGTCTC 17
sgRNA-U16 AAGTATAATAGTAAAAATTG 18
sgRNA-U17 TTTTGTTATACACAATGTTA 19
sgRNA-U18 TTAACATTGTGTATAACAAA 20
sgRNA-U19 ATTCCAAATAGTAATGTACT 21
sgRNA-U20 CTGCCTAGTACATTACTATT 22
sgRNA-U21 AAATAAAAGAAAATAAAGTA 23
sgRNA-U22 AAAATAAAAGAAAATAAAGT 24
sgRNA-U23 AACTTAACCATAGAAAAGAA 25
sgRNA-U24 TAATCATTATACATATTTAT 26
sgRNA-U25 GTACACATATTGACCAAATC 27
sgRNA-U26 TACACATATTGACCAAATCA 28
sgRNA-U27 AATGCAAAATTACCCTGATT 29
sgRNA-U28 GCCCTGAAAGAAAGAGATTA 30
sgRNA-U29 TGCCCTGAAAGAAAGAGATT 31
sgRNA-U30 TTCCCTAATCTCTTTCTTTC 32
sgRNA-U31 TCCCTAATCTCTTTCTTTCA 33
sgRNA-U32 TTCTTTAGAATGGTGCAAAG 34
sgRNA-U33 ATCACTGTTATTCTTTAGAA 35
sgRNA-U34 AGAATAACAGTGATAATTTC 36
sgRNA-U35 GAATAACAGTGATAATTTCT 37
sgRNA-D1 GATTATTCTGAGTCCAAGCT 38
sgRNA-D2 CAGCTCCTGGGCAACGTGCT 39
sgRNA-D3 AGCTGTGGGAGGAAGATAAG 40
sgRNA-D4 AATAAAAGCAGAATGGTAGC 41
sgRNA-D5 ACCATTCTGCTTTTATTTTA 42
sgRNA-D6 TTCTGCTTTTATTTTATGGT 43
sgRNA-D7 TCTGCTTTTATTTTATGGTT 44
sgRNA-D8 TTTATTTTATGGTTGGGATA 45
sgRNA-D9 TTTTATGGTTGGGATAAGGC 46
sgRNA-D10 ATTAGCAAAAGGGCCTAGCT 47
sgRNA-D11 AGCACGTTGCCCAGGAGCTG 48
sgRNA-D12 TATGAACATGATTAGCAAAA 49
sgRNA-D13 GTATGAACATGATTAGCAAA 50
sgRNA-D14 ACCATAAAATAAAAGCAGAA 51
sgRNA-D15 CGTTGCCCAGGAGCTGTGGG 52
sgRNA-D16 GCACGTTGCCCAGGAGCTGT 53
2、sgRNA的制备
化学修饰合成表1中共51条sgRNA-U和sgRNA-D。
3、sgRNA以及Cas9蛋白复合物的电转
将经由化学修饰合成的sgRNA-U或sgRNA-D及Cas9蛋白按一定比例混合后室温孵育10min，同时电转不同sgRNA-U和sgRNA-D组合(以下简称为sg-U+D)至β-地贫IVS2-654 C>T缺陷型造血干细胞，并以未加入任何RNP的β-地贫IVS2-654 C>T缺陷型造血干细胞为空白对照，按电转试剂盒比例混合电转液，电转细胞数量不超过10 5个，细胞离心后电转液重悬细胞并与上述孵育的sgRNA和Cas9蛋白复合物轻轻混匀后转移至电转杯，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℃、5％CO 2培养箱中分化培养，完成缺陷型造血干细胞致病突变位点的破坏和删除。
上述电转sgRNA-U和sgRNA-D的组合如下：
sgRNA-U1(或sgRNA-U16或sgRNA-U32)和sgRNA-D1、
sgRNA-U2(或sgRNA-U17或sgRNA-U33)和sgRNA-D2、
sgRNA-U2(或sgRNA-U18或sgRNA-U34)和sgRNA-D3、
sgRNA-U3(或sgRNA-U19或sgRNA-U35)和sgRNA-D4、
sgRNA-U4(或sgRNA-U20)和sgRNA-D5、
sgRNA-U5(或sgRNA-U21)和sgRNA-D6、
sgRNA-U6(或sgRNA-U22)和sgRNA-D7、
sgRNA-U7(或sgRNA-U23)和sgRNA-D8、
sgRNA-U8(或sgRNA-U24)和sgRNA-D9、
sgRNA-U9(或sgRNA-U25)和sgRNA-D10、
sgRNA-U10(或sgRNA-U26)和sgRNA-D11、
sgRNA-U11(或sgRNA-U27)和sgRNA-D12、
sgRNA-U12(或sgRNA-U28)和sgRNA-D13、
sgRNA-U13(或sgRNA-U29)和sgRNA-D14、
sgRNA-U14(或sgRNA-U30)和sgRNA-D15、
sgRNA-U15(或sgRNA-U31)和sgRNA-D16。
二、鉴定结果
1、基因组DNA的突变鉴定
方法：将步骤一中的步骤3得到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扩增后凝胶电泳检测扩增产物，鉴定突变效率及 DNA片段的缺失状况，剩余细胞继续在EDM-1培养基中分化至第7天。
其中，PCR使用的引物序列如下：
654-U+D-F：5’-GCTTCTGACACAACTGTGTTC-3’(SEQ ID No.68)；
654-U+D-R：5’-CTTTGCCAAAGTGATGGGCCA-3’(SEQ ID No.69)。
结果：与未导入任何RNP的空白对照相比，电转sg-U+D后IVS2-654病人来源造血干细胞在目标位点存在大片段DNA的缺失和删除，其中，电转sg-U+D(具体为sgRNA-U1和sgRNA-D1)的结果如图7所示。
2、EDM-2、EDM-3分化培养
方法：待凝胶电泳确定目标位点删除成功后即可在EDM-2培养基中继续分化。此阶段细胞可大量扩增，可提前冻存部分细胞待用，待EDM-2分化阶段结束，将剩余细胞至EDM-3中继续分化，待分化结束后提取RNA反转成cDNA，用于下述步骤3-5的检测。
3、PCR扩增HBB的外显子
方法：使用引物654-exon1-F和654-exon3-R PCR扩增分化后的外显子，验证致病位点突变后是否能正常剪接。
结果：与未导入任何RNP的空白对照相比，电转sg-U+D后IVS2-654病人来源造血干细胞的异常剪接与正常剪接条带强度比值显著降低，即致病位点经编辑后内含子基本恢复正常剪接，其中，电转sg-U+D(具体为sgRNA-U1和sgRNA-D1)的结果如图8所示。
4、PCR扩增HBB的CDS区
方法：患者造血干细胞致病位点编辑并红细胞分化后，从完整cDNA中PCR扩增HBB编码区序列并进行Sanger测序；其中，所用引物为HBB-CDS-F：和HBB-CDS-R。
结果：与未导入任何RNP的空白对照相比，电转sg-U+D后IVS2-654病人来源造血干细胞HBB基因恢复正常编码功能且接近健康细胞，突变效率可高达95％，其中，电转sg-U+D(具体为sgRNA-U1和sgRNA-D1)的结果如图9所示。
5、qPCR检测
方法：qPCR分析致病位点突变后的造血干细胞中珠蛋白含量变化，其中，qPCR所用引物为：HBA-S、HBA-A、HBB_e2-e3、R_HBB_exon3、HBG-S和HBG-AS。
结果：经sgRNA-U和sgRNA-D编辑的患者造血干细胞，HBB和HBA的mRNA比值接近60％，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，其中，电转sg-U+D(具体为sgRNA-U1和sgRNA-D1)的结果如图10所示。
实施例3、对HUDEP-2细胞中IVS2-654位点所在的HBB第二内含子的编辑
本实施例中使用的细胞为HUDEP-2细胞(Human Umbilical Cord Blood  Derived Erythoid Progenitor-2，人脐带血来源的红系祖细胞)。
一、编辑方法
1、sgRNA的制备
化学修饰合成表1中的sgRNA-U1、sgRNA-U2、sgRNA-U3，sgRNA-D1、sgRNA-D2、sgRNA-D3、sgRNA-D4。
2、sgRNA以及Cas9蛋白复合物的电转
按照实施例2步骤一中的步骤3方法进行，不同之处在于，共设置如下不同处理：
空白对照：未加入任何RNP的HUDEP-2细胞；
处理1：电转sgRNA-U1和sgRNA-D1的HUDEP-2细胞；
处理2：电转sgRNA-U2和sgRNA-D2的HUDEP-2细胞；
处理3：电转sgRNA-U2和sgRNA-D3的HUDEP-2细胞；
处理4：电转sgRNA-U3和sgRNA-D4的HUDEP-2细胞。
二、鉴定结果
1、基因组DNA的突变鉴定
方法：按照实施例2步骤二中的步骤1方法进行。
结果：如图11所示，与未导入任何RNP的空白对照相比，经过处理1-4编辑后造成HBB第二内含子基因区不同长度的DNA删除，且该DNA删除的长度与预期相同。
2、EDM-2、EDM-3分化培养
按照实施例2步骤二中的步骤2方法进行。
3、PCR扩增HBB的外显子
方法：按照实施例2步骤二中的步骤3方法进行。
结果：如图12所示，在HUDEP-2细胞中，经处理1-4编辑后IVS2-654至HBB第二内含子基因区两端分别靶向编辑并红细胞分化后cDNA的样品剪接情况全部为正常状态。
本实施例进一步验证了在HBB第二内含子中联合使用致病位点上游sgRNA和下游sgRNA两条sgRNA共同靶向切割，造成包括致病位点在内大片段DNA删除，不影响HBB的表达，具有安全性和可行性。

Claims (14)

  1. 一种针对细胞中由于HBB(β-珠蛋白基因)IVS2-654 C>T突变导致内含子异常剪接的修复方法，所述IVS2-654 C>T的突变引入额外的剪接供***点导致内含子异常剪接，其特征在于，所述方法包括利用CRISPR-Cas9***对HBB进行基因编辑以失活或删除所述额外的剪接供***点的步骤；
    所述CRISPR-Cas9***包括Cas9和至少一条靶向目标序列的sgRNA。
  2. 根据权利要求1所述的修复方法，其特征在于，所述sgRNA的靶向位点在IVS2-654 C>T位点的上游20bp至IVS2-654 C>T位点的下游70bp范围内。
  3. 根据权利要求2所述的修复方法，其特征在于，所述sgRNA包括sgRNA-1；所述sgRNA-1的靶向序列为SEQ ID No.1所示序列；优选的，所述sgRNA包括碱基的化学修饰。
  4. 根据权利要求3所述的修复方法，其特征在于，所述sgRNA还包括sgRNA-2；所述sgRNA-2的靶向序列为SEQ ID No.2所示序列；优选的，所述sgRNA包括碱基的化学修饰。
  5. 根据权利要求1所述的修复方法，其特征在于，所述sgRNA包括sgRNA-U和sgRNA-D，
    所述sgRNA-U的靶向位点在IVS2-654 C>T位点的上游21bp至IVS2-654 C>T位点所在的HBB基因第二内含子起始位点范围内，
    所述sgRNA-D的靶向位点在IVS2-654 C>T位点的下游71bp至IVS2-654 C>T位点所在的HBB基因第二内含子终止位点范围内。
  6. 根据权利要求5所述的修复方法，其特征在于，所述sgRNA-U的靶向序列为SEQ ID No.3-37所示序列中的任一种或任两种以上；优选的，SEQ ID No.3、4、和/或5所示序列，更优选的，所述sgRNA-U包括碱基的化学修饰。
  7. 根据权利要求5或6所述的修复方法，其特征在于，所述sgRNA-D的靶向序列为SEQ ID No.38-53所示序列中的任一种或任两种以上；优选的，SEQ ID No.38、39、40和/或41所示序列；更优选的，所述sgRNA-D包括碱基的化学修饰。
  8. 根据权利要求1-7中任一所述的修复方法，其特征在于，所述额外的剪接供***点的序列为AAGGTAATA。
  9. 根据权利要求1-8中任一所述的修复方法，其特征在于，采用电转化的方法向细胞中引入包含Cas9和sgRNA的复合物。
  10. 根据权利要求1-9中任一所述的修复方法，其特征在于，所述细胞为造血干细胞或红系祖细胞，
    优选的，所述造血干细胞为CD34 +的造血干祖细胞，所述红系祖细胞为HUDEP-2。
  11. 一种用于修复由HBB(β-珠蛋白基因)IVS2-654 C>T突变导致的内含子异常剪接的sgRNA，
    所述sgRNA包括sgRNA-1，所述sgRNA-1的靶向序列为SEQ ID No.1所示序列；优选的，所述sgRNA还包括sgRNA-2，所述sgRNA-2的靶向序列为SEQ  ID No.2所示序列；或，
    所述sgRNA包括sgRNA-U和sgRNA-D，
    所述sgRNA-U的靶向位点在IVS2-654 C>T位点的上游21bp至IVS2-654 C>T位点所在的HBB基因第二内含子起始位点范围内；
    所述sgRNA-D的靶向位点在IVS2-654 C>T位点的下游71bp至IVS2-654 C>T位点所在的HBB基因第二内含子终止位点范围内。
  12. 根据权利要求11所述的sgRNA，其特征在于，所述sgRNA-U的靶向序列为SEQ ID No.3-37所示序列中的任一种或任两种以上；优选的，SEQ ID No.3、4、和/或5所示序列，更优选的，所述sgRNA-U包括碱基的化学修饰；
    所述sgRNA-D的靶向序列为SEQ ID No.38-53所示序列中的任一种或任两种以上；优选的，SEQ ID No.38、39、40和/或41所示序列；更优选的，所述sgRNA-D包括碱基的化学修饰。
  13. 权利要求11或12所述的sgRNA在修复细胞中由HBB(β-珠蛋白基因)IVS2-654 C>T突变导致的内含子异常剪接中的应用。
  14. 根据权利要求13所述的应用，其特征在于，所述细胞为造血干细胞或红系祖细胞，
    优选的，所述造血干细胞为CD34 +的造血干祖细胞，所述红系祖细胞为HUDEP-2。
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