WO2018227755A1 - 用于特异性修复人hbb基因突变的碱基编辑系统、方法、试剂盒及其应用 - Google Patents

用于特异性修复人hbb基因突变的碱基编辑系统、方法、试剂盒及其应用 Download PDF

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WO2018227755A1
WO2018227755A1 PCT/CN2017/098360 CN2017098360W WO2018227755A1 WO 2018227755 A1 WO2018227755 A1 WO 2018227755A1 CN 2017098360 W CN2017098360 W CN 2017098360W WO 2018227755 A1 WO2018227755 A1 WO 2018227755A1
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grna
sequence
base
hbb
base editing
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黄军就
松阳洲
梁普平
孙宏伟
张曦亚
孙英
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中山大学
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Definitions

  • the invention relates to the field of gene detection and gene modification, and relates to a base editing system, method, kit and application thereof for specifically repairing a human HBB gene mutation.
  • CRISPR-associated CRISPR-associated, CRISPR-Cas
  • CRISPR-associated CRISPR-associated, CRISPR-Cas
  • Invasions such as phage viruses and foreign plasmids.
  • it provides acquired immunity to bacteria: this is similar to mammalian acquired immunity, when the bacteria are invaded by viruses or foreign plasmids, they produce corresponding "memory", which can resist their re-invasion.
  • the CRISPR/Cas system recognizes foreign DNA or RNA and severs them to silence expression of foreign genes. Thanks to this precise targeting function, the CRISPR/Cas system was developed as an efficient gene editing tool.
  • the CRISPR-Cas system is divided into two categories.
  • the first major class of CRISPR-Cas systems functions as a multi-subunit effector complex; the second class is played by a single effector protein (such as Cas9, Cpf1, C2c1, etc.).
  • a single effector protein such as Cas9, Cpf1, C2c1, etc.
  • Cas9, Cpf1, and C2c1 all have RNA-mediated DNA endonuclease activity.
  • Cas9 and Cpf1 proteins are widely used as genome editing tools, which overcomes the shortcomings of traditional gene editing techniques, such as cumbersome, time-consuming and inefficient, and meets most of them with less components, convenient operation and high efficiency.
  • the genetic editing needs of the field have potential and enormous clinical application value.
  • CRISPR/Cas9 is a complex with endonuclease activity that recognizes specific DNA sequences and performs site-specific cleavage resulting in double-stranded DNA breaks (DSB), in the absence of a template.
  • Non-homologous end joining (NHEJ) resulting in a frameshift mutation, leading to gene knockout.
  • CRISPR/Cas9 is a "Zinc Finger Endonuclease (ZFN)", a “like transcriptional activator”
  • ZFN Zinc Finger Endonuclease
  • TALEN effector nuclease
  • the CRISPR/Cas9 system is a powerful tool for gene editing, allowing precise editing of genes. It mainly consists of Cas9 and RNA. Cas9 targeted cleavage of DNA is achieved by the principle of complementary recognition of two small RNAs, cryRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) and target sequences.
  • CRISPR RNA cryRNA
  • tracrRNA trans-activating crRNA
  • target sequences target sequences.
  • the two small RNAs have now been fused into one piece of RNA (chimeric RNA), abbreviated as sgRNA (small guide RNA). Both the dimer of crRNA-tracrRNA and sgRNA can be used as guide RNA (gRNA) to mediate the targeting of the CRISPR/Cas9 system.
  • gRNA guide RNA
  • CRISPR/Cas9-mediated DNA site-directed cleavage also requires the help of a PAM (Protospace multiples) sequence that is relatively conserved for each CRISPR/Cas system near the target site to recognize target DNA complementary to the gRNA leader sequence.
  • PAM Protospace multiples
  • the Cas9 protein will look for the target DNA under the guidance of the gRNA and cleave the target DNA.
  • the organism itself has a response mechanism for DNA damage repair, which will connect the sequences at the upstream and downstream ends of the break, thereby achieving knockout of the target gene in the cell.
  • a repaired template plasmid (donor DNA molecule) is introduced into the cell on this basis, the cell will accurately introduce site-directed mutagenesis or site-directed insertion of the foreign fragment by homologous recombination during the repair process according to the template provided.
  • CRISPR/Cas technology has been widely used. In addition to basic editing methods such as gene knockout and gene replacement, it can also be used for gene activation, disease model building, and even gene therapy.
  • CRISPR/Cas9 UGI fusion protein (bases editing enzyme) and two portions gRNA constituents precise gene-editing technique 5-7.
  • Cytidine deaminase Cas9 in the fusion protein of Cas9: UGI binds to gRNA and utilizes the sequence of gRNA to target the fusion protein to the target DNA through base pairing. Then, the base editing enzyme utilizes Cytidine.
  • deaminase converts C (cytosine) in positions 4-8 of target DNAd to uracil (Uridine, U)
  • UGI is a uracil DNA glycosylase inhibitor (Uracil DNA) Glycosylase inhibitor (UGI), which inhibits U excision, causes U to pair with A (adenine, Adenine) during replication, and then replicates through DNA, making U a T (Thymine, thymine), which will eventually C is converted to T 5,6,8 .
  • Base-editing technology-mediated DNA site-directed mutagenesis is more than 100-fold more efficient than mutations mediated by homologous recombination techniques, and can achieve 100% site-directed mutagenesis efficiency in some mouse fertilized eggs.
  • the base editing enzyme is named Base editor 2 (BE2) or Base editor 3 (BE3), wherein BE2 can not cleave double-stranded target DNA at all, and BE3 can double-strand The target strand in the target DNA is cleaved.
  • the base editing system can specifically and accurately target the target base of the target gene is whether the base editing system can Prerequisite for specific site-directed mutagenesis of genes.
  • the specificity of the base editing system is mainly determined by Cas9 and gRNA. Specificity can be improved by site-directed mutagenesis of Cas9, thereby reducing the non-specific interaction of Cas9 with the target strand or the non-target strand phosphate backbone of the double-stranded target DNA.
  • designing, preparing, and screening gRNAs that are specifically complementary to the target DNA are also key factors in determining the efficiency and specificity of the base editing system.
  • Thalassemia is a genetic disease caused by abnormal synthesis of erythrocyte hemoglobulin.
  • the patient's red blood cells are smaller and more fragile than normal red blood cells and are very prone to rupture, leading to hemolytic anemia.
  • its oxygen carrying capacity is also weaker than normal cells, resulting in hypoxia in various tissues of the body.
  • Adult blood globulin contains three types of Hb A2 (2.5%), Hb F (0.5%) and Hb A (97%).
  • Hb A immunoglobulin protein consists of two ⁇ subunits and two ⁇ subunits globulin 1. According to the different subunits, thalassemia can be divided into alpha thalassemia and beta thalassemia.
  • beta thalassemia is a common monogenic genetic disease caused by mutation of blood globulin ⁇ globulin subunit, and its coding gene is HBB (human ⁇ globin) 1 .
  • HBB human ⁇ globin
  • Mutation of HBB reduces or becomes unstable of ⁇ -globulin synthesis, resulting in an imbalance of the ratio of ⁇ -globulin and ⁇ -globulin in blood globulin, and a decrease in blood globulin, which in turn affects the maturation of red blood cells, resulting in a decrease in the number of mature red blood cells.
  • alpha globulin and its degradation products accumulated in red blood cells cause red blood cell lysis. The two factors are superimposed together to cause anemia 2 .
  • beta thalassemia can be divided into: micro beta thalassemia, moderate beta thalassemia and severe beta thalassemia.
  • Patients with severe beta thalassemia usually develop severe anemia, stagnant growth, skeletal abnormalities, and hepatosplenomegaly around the age of one.
  • iron ions in hemoglobin are released, resulting in excessive concentration of iron ions in the blood, resulting in damage to multiple organs such as the liver and heart. Long-term liver and heart damage can lead to cirrhosis and heart failure, which is life-threatening 3 .
  • patients with severe beta thalassemia need to be treated with long-term blood transfusion or bone marrow transplantation.
  • long-term blood transfusion to patients with blood transfusion will lead to high blood iron concentration, and the adjuvant treatment of iron ion chelating agents is also a burden on the patient's body. Therefore, in patients receiving chronic transfusion therapy, 50% of patients will be dead before the age of 35 4.
  • Patients with moderate beta thalassemia have a milder proportion of the disease, usually with moderate or severe anemia in their early or late years, and usually do not require transfusion therapy.
  • Patients with micro-thalassemia are usually not anemia or mild anemia.
  • Beta thalassemia is very common in the Mediterranean region, Africa, the Middle East, the Indian subcontinent, Southeast Asia, and southern China. In some regions, the frequency of HBB mutations is as high as 10%. In different regions, there are some differences in HBB mutation sites and their frequencies. 2 .
  • the mutations of HBB can be divided into two categories: the first is a ⁇ 0 mutation that causes ⁇ globulin to fail to synthesize, and the second is a ⁇ + mutation that causes a decrease in the amount of ⁇ globulin synthesis.
  • HBB: c.-79A>G and HBB: c.-78A>G mutations are all ⁇ + mutations. The mutated heterozygotes usually do not develop disease, or only mild anemia.
  • Clinical treatment options for beta thalassemia include: transfusion therapy and bone marrow transplantation.
  • Long-term blood transfusion treatment is a great burden on the patient's body and mind, which seriously affects the patient's quality of life.
  • For bone marrow transplantation it is necessary to find a donor with a successful leukocyte antigen (HLA) matching, which limits its application range.
  • HLA leukocyte antigen
  • Traditional gene therapy improves the patient's blood globulin by extracting the patient's hematopoietic stem cell (HSC) or bone marrow cells and introducing the beta globulin or gamma globulin coding sequence into the HSC via retrovirus or lentiviral vector.
  • base editing systems have been applied to single-base gene editing 8-10 for plants, yeast, human cells, and mouse embryos, but whether base-editing systems can be used to repair HBB: c.-79A>G and HBB: The c.-78A>G mutation and treatment of beta thalassemia caused by the above mutations at the cellular level remains unknown.
  • the present invention provides a base editing system, method, kit and application thereof for specifically repairing a human HBB gene mutation.
  • a first aspect of the present invention provides a base editing system for specifically repairing a human HBB gene mutation, comprising: a base editing enzyme and a gRNA, the base editing enzyme being a fusion protein, the fusion protein Including the effector domain of the CRISPR/Cas system, the Cytidine deaminase domain, and the Uracil DNA glycosylase inhibitor (UGI) domain, including but not limited to suppuration Sp-gRNA of Streptococcus pyogenes Cas9 (SpCas9), Sa-gRNA of Staphylococcus aureus Cas9 (SaCas9), Cj-gRNA of Campylobacter Cas9 (Campylobacter jejuni Cas9, CjCas9), St-gRNA of Streptococcus thermophilus Cas9 (StCas9), Nm-gRNA of Neisseria meningitidis Cas9, NmCas9, Lb-g
  • the gRNA comprises from about 15 to 100 nucleotides and further comprises a leader sequence consisting of at least 12 contiguous nucleotides complementary to the target DNA sequence.
  • the sequence in which the gRNA is complementary to the target DNA comprises one or more of the nucleotide sequences set forth in SEQ ID NO. 1 - SEQ ID NO.
  • the target DNA sequence complementary to the gRNA leader sequence is a human genomic DNA sequence adjacent to the HBB: c.-79A>G and HBB:c.-78A>G mutations.
  • the gRNA is one of the following 1) or 2):
  • a crRNA and a tracrRNA comprising a crRNA and a tracrRNA, wherein a partial sequence of the crRNA is complementary to a partial sequence of the tracrRNA and comprises a dimer, wherein the leader sequence of the crRNA comprises the nucleotide sequence of SEQ ID NO. 1 - SEQ ID NO.
  • a chimeric single-stranded sgRNA wherein the sgRNA is fused by a crRNA and a tracrRNA, and the leader sequence of the sgRNA comprises one or more of the nucleotide sequences shown in SEQ ID NO. 1 - SEQ ID NO.
  • the gRNA comprises a backbone sequence in addition to a leader sequence complementary to the target DNA sequence.
  • the gRNA can be in the form of a dimer consisting of crRNA and tracrRNA, or an artificially engineered chimeric single-stranded sgRNA fused from crRNA and tracrRNA.
  • RNA molecule of CRISPR RNA is formed by base pairing with a part of its sequence (3' end) and an RNA molecule called tracrRNA (trans-activating crRNA).
  • Chimeric RNA ie tracrRNA/crRNA dimer
  • the chimeric RNA is able to guide
  • the base editing enzyme is bound to the target DNA sequence and edited for editing.
  • tracrRNA and crRNA can also be fused together to form a single guide RNA (sgRNA), which is a chimeric single-stranded sgRNA according to the present invention.
  • sgRNA single guide RNA
  • the chimeric single-stranded sgRNA is capable of directing the base editing enzyme to bind to the target DNA sequence and editing the clip.
  • the effector domain of the CRISPR/Cas system is an effector protein of the CRISPR/Cas system having no cleavage activity or only single-strand cleavage activity.
  • the effector protein of the CRISPR/Cas system includes, but is not limited to, Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Campylobacter jejuni Cas9 (Campylobacter jejuni Cas9, CjCas9), Streptococcus thermophilus Cas9 (StCas9), Cas9 (Neisseria meningitidis Cas9, NmCas9), Cpf1 (Lachnospiraceae Cpf1, LbCpf1), Acetobacter Cpf1 Recombinant or mutant of an effector protein such as (Acidaminococcus C
  • the effector domain of the CRISPR/Cas system includes, but is not limited to, the recombinant Cas9 protein disclosed in International Publication No. PCT/US2016/058345 or CT/US2016/058344 International Publication.
  • amino acid sequences set forth in SEQ ID NO. 9-262 as disclosed in the International Publication of PCT/US2016/058345 are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, 99. % or 99.5% consistent and not identical to the naturally occurring Cas9 protein;
  • any one of SEQ ID NO. 9-262 contains one, two, three at least in the 262, 267, 294, 405, 409, 480, 543, 694, 1219, 1224, 1256, 1362 amino acid positions. , four, five, six or seven mutations.”
  • the base-editing enzyme includes, but is not limited to, a base-editing fusion protein disclosed in International Publication No.: PCT/US2016/058345 or CT/US2016/058344 International Publication (ie, the present invention The base editing enzyme described on the one hand).
  • the base-editing fusion proteins disclosed in the prior art i.e., the base-editing enzymes described in the first aspect of the invention
  • the cytosine deaminase domain includes, but is not limited to, APOBEC1 in rat, APOBEC3 in mouse, APOBEC1 in human, APOBEC3A in human, APOBEC3B in human, APOBEC3C in human, human APOBEC3D, human APOBEC3F, human APOBEC3G, human APOBEC3H, human AID, sputum CDA1 and other cytosine deaminase.
  • the uracil DNA glycosylase inhibitory domain includes, but is not limited to, a uracil DNA glycosylase inhibitor (UGI) of Bacillus subtilis phage PBS1 and a uracil of Bacillus subtilis PBS2 DNA glycosylase inhibitor (UGI).
  • the amino acid sequence of the base-editing enzyme comprises, but is not limited to, at least 80%, 85%, 90%, 92%, 95%, 96 of the amino acid sequence shown in SEQ ID NO. 94-105. %, 97%, 98%, 99%, or 99.5% consistent sequence.
  • amino acid sequences shown in SEQ ID NO. 94-105 correspond to BE2, BE3, HF1-BE2, HF1-BE3, HF2-BE2, HF2-BE3, VQR-HF1-BE2, VQR-HF1-BE3, respectively.
  • the coding nucleotide sequence of the above amino acid sequence by means of conventional gene synthesis and site-directed mutagenesis in the art.
  • the coding DNA sequence of the amino acid sequence of SEQ ID NO. 94-105 was synthesized and cloned into pcDNA3.1(-) vector (Invitorgen) by digestion and ligation with XbaI and BamHI to prepare an expression system.
  • pcDNA3.1(-) vector Invitorgen
  • the gRNA consisting of the leader sequence and the backbone sequence may be a coding corresponding to the gRNA.
  • the DNA sequence of the code is cloned into a vector containing a T7 or Sp6 promoter (such as pDR274, Addgene), or directly added to the T7 or Sp6 promoter at the front end of the corresponding DNA encoding DNA by PCR, annealing, synthesis, etc., and can be transcribed.
  • the transcript gRNA sequence was obtained.
  • the gRNA can be directly synthesized, wherein the gRNA can be a common gRNA or a chemically modified gRNA, and the chemical modification includes modification of the RNA backbone and the base.
  • the gRNA can specifically recognize the HBB mutant gene by fully complementary or substantially complementary, or with a certain percentage of complementation (in the present embodiment, the "HBB mutant gene” and the “target nucleotide sequence” "Can be interchanged.”
  • the gRNAs provided herein are sufficiently complementary to the target nucleotide sequence to hybridize to the target nucleotide sequence and direct the specific binding of the base editing enzyme to the target nucleotide sequence.
  • the degree of complementarity between the gRNA and its corresponding target nucleotide sequence is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more.
  • complementary refers to the formation of one or more hydrogen bonds between a nucleic acid and another nucleic acid sequence by means of conventional Watson-Crick base pairing or other non-conventional types.
  • “Complementary percentage” means the percentage of residues in a nucleic acid molecule that can form a hydrogen bond (eg, Watson-Crick base pairing) with a second nucleic acid sequence (eg, 5, 6, 7 out of 10) 8, 8, 9, 10 are 50%, 60%, 70%, 80%, 90%, and 100% complementary).
  • “Completely complementary” means that all contiguous residues of one nucleic acid sequence form a hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • substantially complementary means having 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, At least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98 in the region of 30, 35, 40, 45, 50 or more nucleotides %, 99%, or 100% complementarity.
  • the gRNA is a mature gRNA sequence consisting of a leader sequence and a backbone sequence.
  • the leader sequence is one or more of the nucleotide sequences set forth in SEQ ID NO. 1 - SEQ ID NO. 93 of Table 1.
  • the gRNA backbone sequence is conventionally used by those skilled in the art, and the backbone sequence may be a dimer composed of crRNA and tracrRNA (trans-activating crRNA), or may be artificially modified by crRNA and A chimeric single-stranded sgRNA fused with tracrRNA.
  • the backbone sequence of the gRNA may be a sequence currently used conventionally, such as GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC, or some mutants constructed on the basis of these, the mutants are 50%, 60%, 75%, 80%, and the sequences currently used conventionally. Similarity of 85%, 90%, 95%, 97.5%, 99% or higher.
  • the target nucleotide specifically recognizable by the gRNA sequence may include a disease associated with HBB disease Gene or polynucleotide.
  • a "disease-associated" gene or polynucleotide refers to any gene or polynucleoside that produces a transcriptional or translational product at an abnormal level or in an abnormal form in a cell derived from a disease-affected tissue as compared to a non-disease-controlled tissue or cell. acid. Where the altered expression is associated with the appearance and/or progression of the disease, it can be a gene that is expressed at an abnormally high level; it can be a gene that is expressed at an abnormally low level.
  • a disease-associated gene also refers to a gene having one or more mutations or a genetic variation that is directly responsible for or incompatible with one or more genes responsible for the etiology of the disease. The transcribed or translated product may be known or unknown and may be at normal or abnormal levels.
  • the specifically repaired human HBB gene mutations include, but are not limited to, human HBB: c.-79A>G and HBB: c.-78A>G mutation.
  • target nucleotide refers to a ribonucleotide or an analog thereof.
  • a “target nucleotide” may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs.
  • a first linker peptide is further linked between an effector domain of the CRISPR/Cas system and a Cytidine deaminase domain.
  • the effector domain of the CRISPR/Cas system and the Uracil DNA glycosylase inhibitor (UGI) domain are A second linker peptide is also attached.
  • the first linker peptide is (GGGS)n, (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, (SGGS)n, SGSETPGTSESATPES or XP)n
  • n is a natural number from 1 to 30 and X is any one of amino acids.
  • the first linker peptide is an XTEN-linked polypeptide.
  • the second linker peptide is one of (GGGGS)n, (G)n, (EAAAK)n, (GGS)n, (SGGS)n, SGSETPGTSESATPES, SGGS or XP)n
  • n is a natural number from 1 to 30, and X is any one of amino acids.
  • the second linker peptide is an XTEN-linked polypeptide.
  • the first linker peptide and the second linker peptide may be the same or different.
  • the base-editing enzyme fusion protein further comprises other linking peptides optionally between any two domains, and the linking peptides include, but are not limited to, a protein tag, a reporter gene sequence, and a protein domain of one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repressor activity, transcription release factor activity, histone modification activity , RNA cleavage activity and nucleic acid binding activity.
  • Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza virus hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag .
  • reporter genes include, but are not limited to, Glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), ⁇ -galactosidase, ⁇ -glucuronidase, fluorescein Photozyme, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent protein including blue fluorescent protein (BFP).
  • Glutathione-S-transferase GST
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • ⁇ -galactosidase ⁇ -glucuronidase
  • fluorescein Photozyme green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent protein including blue fluorescent protein (BFP).
  • the base-editing enzyme fusion protein may further comprise a protein or protein fragment, the protein or protein fragment binding to the DNA molecule or binding to other cellular molecules, including, but not limited to, Maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusion, GAL4 DNA binding domain fusion, and herpes simplex virus (HSV) BP16 protein fusion.
  • MBP Maltose binding protein
  • DBD Lex A DNA binding domain
  • GAL4 DNA binding domain fusion GAL4 DNA binding domain fusion
  • HSV herpes simplex virus
  • polypeptide refers to a polymer of amino acids of any length.
  • the polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified; such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other modification, such as binding to a labeled component of a detection molecule .
  • a second aspect of the invention provides a non-naturally occurring or engineered composition which is one or more of the following 1) to 6):
  • the component I comprises a first regulatory element, and a coding sequence encoding a base-editing enzyme as described in the first aspect, operably linked to the first regulatory element; the component II comprising a second regulatory element And a coding sequence encoding the gRNA of the first aspect, operably linked to the second regulatory element; wherein components I and II are on the same or different vectors;
  • An mRNA comprising a base-editing enzyme according to the first aspect, and a vector comprising a second regulatory element, and a gRNA encoding according to the first aspect, operably linked to the second regulatory element Coding sequence
  • a protein comprising a base-editing enzyme according to the first aspect, and a vector, the vector comprising a second regulatory element, and a gRNA as described in the first aspect, operably linked to the second regulatory element Coding sequence
  • a protein comprising the base editing enzyme of the first aspect and the gRNA of the first aspect.
  • the base-editing enzyme of the first aspect may be in the form of DNA, RNA or protein; the gRNA may be in the form of DNA or RNA.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it is linked.
  • Vectors include, but are not limited to, single-stranded, double-stranded, or partially double-stranded nucleic acid molecules; nucleic acid molecules comprising one or more free ends, no free ends (eg, circular); including DNA, RNA, or both Nucleic acid molecules; and in the field A wide variety of other polynucleotides are known.
  • plasmid refers to a circular double stranded DNA loop in which additional DNA fragments can be inserted, for example, by standard molecular cloning techniques.
  • a viral vector in which a virus-derived DNA or RNA sequence is present for packaging a virus (eg, retrovirus, replication-defective retrovirus, adenovirus, replication-defective adenovirus) And the vector of adeno-associated virus).
  • the viral vector also comprises a polynucleotide carried by a virus for transfection into a host cell.
  • Certain vectors e.g., bacterial vectors having bacterial origins of replication and episomal mammalian vectors
  • vectors e.g., non-episomal mammalian vectors
  • Other vectors are integrated into the genome of the host cell upon introduction into the host cell and thereby replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "expression vectors.”
  • Common expression vectors used in recombinant DNA techniques are typically in the form of plasmids.
  • operably linked is intended to mean that the nucleotide sequence is linked to one or more regulatory elements in a manner that allows expression of the nucleotide sequence (optionally, the vector is in a
  • the nucleotide sequence can be expressed in an in vitro transcription/translation system; alternatively, the nucleotide sequence can be expressed when the vector is introduced into a host cell).
  • the term "expression” refers to the process of transcription from a DNA template into a polynucleotide (eg, transcription into mRNA or other RNA transcript) and/or the subsequent transcription of the transcribed mRNA into a peptide, polypeptide or protein. .
  • the transcripts and encoded polypeptides may be collectively referred to as "gene products.” If the polynucleotide is derived from genomic DNA, expression can include splicing of mRNA in eukaryotic cells.
  • non-naturally occurring or “engineered” as used herein, when used interchangeably, when referring to a nucleic acid molecule or polypeptide, means that the nucleic acid molecule or polypeptide is at least substantially from them in nature or as found in nature. At least one other component that is combined with it is freed.
  • the first regulatory element in one embodiment of the invention, the "regulatory element” of the invention may be understood as a "gene expression cassette" comprising one or more pol III promoters. a promoter (eg 1, 2, 3, 4, 5, or more pol III promoter), one or more pol II promoters (eg 1, 2, 3, 4, 5, or more pol II promoters) , one or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof.
  • the first regulatory element may also be a T7 promoter, a Sp6 promoter or the like. Examples of pol III promoters include, but are not limited to, the U6 and H1 promoters.
  • pol II promoters include, but are not limited to, a retroviral Rous sarcoma virus (RSV) LTR promoter (optionally having an RSV enhancer), a cytomegalovirus (CMV) promoter (optionally having a CMV enhancer) [ See, for example, Boshart et al., Cell 41:521-530 (1985), SV40 promoter, dihydrofolate reductase promoter, beta-actin promoter, phosphoglycerol Kinase (PGK) promoter, and EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol Kinase
  • the protein coding sequence is codon optimized for expression in a particular cell, such as a eukaryotic cell.
  • eukaryotic cells may be those of a particular organism or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non-human primate.
  • codon optimization refers to use in the host
  • the codons used more frequently or most frequently in the genes of the cell replace at least one codon of the native sequence (eg, about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50) , or a method in which a plurality of codons simultaneously maintain the native amino acid sequence to modify a nucleic acid sequence to enhance expression in a host cell of interest.
  • Different species exhibit particular preferences for certain codons having a particular amino acid.
  • Sub-preference difference in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example in the Codon Usage Database, and these tables can be adapted in different ways. See, Nakamura Y., et al., "Codon usage tabulated from the international DNA sequence databases: status for the year 2000". Nucl. Acids Res. 28: 292 (2000). Computer algorithms for codon-optimizing specific sequences for expression in specific host cells are also available, such as Gene Forge. (Aptagen; Jacobus, PA), also available.
  • one or more codons in a sequence encoding a CRISPR enzyme eg 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons correspond to the most frequently used codons for a particular amino acid.
  • the second regulatory element comprises, but is not limited to, one or more pol II promoters (eg 1, 2, 3, 4, 5, or more pol II promoters), One or more pol I promoters (eg, 1, 2, 3, 4, 5, or more pol I promoters), or a combination thereof, pol III promoter (including but not limited to U6, H1 promoter) , T7 promoter, Sp6 promoter, etc.
  • the first or second regulatory element further comprises an enhancer, an internal ribosome entry site (IRES), and other expression control elements (eg, a transcription termination signal, such as a polyadenylation signal) And poly U sequences).
  • IRES internal ribosome entry site
  • other expression control elements eg, a transcription termination signal, such as a polyadenylation signal
  • Regulatory elements include those sequences that direct constitutive expression of a nucleotide sequence in many types of host cells, as well as those sequences that direct expression of the nucleotide sequence only in certain host cells (eg, tissue-specific regulatory sequences) .
  • Tissue-specific promoters can primarily direct expression in a desired tissue of interest, such as muscle, neurons, bone, skin, blood, specific organs (eg, liver, pancreas), or specific cell types (eg, Lymphocytes).
  • the regulatory elements may also direct expression in a time-dependent manner (eg, in a cell cycle dependent or developmental stage dependent manner), which may or may not be tissue or cell type specific.
  • a third aspect of the invention provides a method for specifically repairing a human HBB gene mutation in a somatic cell or a human, the method comprising delivering the base editing system of the first aspect or the combination of the second aspect The sequence of the base editing system or composition associated with the human HBB mutant gene is approximated to obtain a repaired human HBB gene.
  • the term "proximating the sequence of the base editing system or composition associated with a human HBB mutated gene” means delivering the component to an in vitro or in vivo environment.
  • an in vivo environment such as intracellular; the term “proximity” means in an in vitro or in vivo environment.
  • Each component can be contacted with a sequence associated with a human HBB mutant gene and, under certain conditions, can occur as would be expected by those skilled in the art.
  • the term "in human body” may be in vitro or in vivo, for example, may be in the human germline; it is noted that on the same day of the present invention
  • the Applicant specifically claims a method for specifically repairing a human HBB gene mutation in the human germline, in particular, in another patent, the human germline includes human germ cells. , fertilized egg or embryo; in the scope of the claims of the third aspect of the present invention, "in the human body” does not include the claims in the claims corresponding to another patent filed on the same day of the present invention. Within the reproductive system").
  • the invention provides a method comprising delivering one or more polynucleotides, one or more vectors, one or more transcripts, and/or one or more to a somatic cell or human Transcribed proteins. In some aspects, the invention further provides cells, fertilized eggs and embryos produced by such methods.
  • the base editing system complex in combination with gRNA is delivered to a somatic cell or human.
  • the base editing system of the first aspect or the composition of the second aspect can be introduced into a human somatic cell or individual using conventional viral and non-viral based gene transfer methods.
  • Non-viral vector delivery systems include DNA plasmids, RNA (such as the transcripts of the vectors described herein), proteins, naked nucleic acids, and nucleic acids complexed with delivery vehicles such as liposomes.
  • Viral vector delivery systems include DNA and RNA viruses that have a free or integrated genome upon delivery to a cell.
  • Non-viral delivery methods for nucleic acids include lipofection, nuclear transfection, microinjection, electroporation, gene guns, viral particles, liposomes, immunoliposomes, polycations or lipids: nucleic acid conjugates, naked DNA, Enhancement uptake of artificial virions and reagents for DNA.
  • Lipid transfection is described in, for example, U.S. Patent Nos. 5,049,386, 4,946,787 and 4,897,355, and the lipofection reagents are commercially available (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids suitable for efficient receptor recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be directed to cells (eg, administered in vitro or ex vivo) or to target tissues (eg, administered in vivo).
  • nucleic acid complexes including targeted liposomes, such as immunolipid complexes
  • lipid nucleic acid complexes, including targeted liposomes, such as immunolipid complexes
  • crystal Science (Science) 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugated Chemistry 5:647-654 (1994); Gao (Gao) et al., Gene Therapy (Gene Therapy) 2: 710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); US Patent Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028 and 4,946,787).
  • the eukaryotic expression vector, prokaryotic expression vector or in vitro transcription vector of the base editing enzyme of the first aspect is prepared; then the eukaryotic expression vector is amplified and prepared without endotoxin.
  • the base editing enzyme coding DNA can also be cloned into an in vitro transcription vector containing a T7 or Sp6 promoter, and the mRNA of the base-editing enzyme can be prepared by in vitro transcription.
  • the gRNA of the first aspect is prepared, and the gRNA can be directly synthesized, or the coding sequence of the gRNA can be cloned into a transcription vector containing a T7 or Sp6 promoter (such as pDR274, from Addgene), or PCR, annealing, synthesis, etc.
  • a transcription vector containing a T7 or Sp6 promoter such as pDR274, from Addgene
  • PCR annealing, synthesis, etc.
  • the pGEM-T-U6-gRNA vector is a method for amplifying the U6 promoter and the gRNA backbone sequence from the pX330 vector (Addgene) by PCR, and cloning the PCR product by TA. It was prepared by ligating into the pGEM-T vector (Promega).
  • the DNA of the base-editing enzyme of the first aspect (transcribed mRNA or translated protein) and the expression vector of gRNA (or transcribed gRNA, or synthetic gRNA) are included but not included. It is limited to liposome transfection, viral infection, electroporation, and microinjection into human somatic cells or humans.
  • the viral vector used for the viral infection includes, but is not limited to, a retrovirus vector, a lentiviral vector, Adenoviral vectors and adenoviruses are first used as viral vectors.
  • human cells include, but are not limited to, bone marrow cells, hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and hematopoietic progenitor cells.
  • HSCs hematopoietic stem cells
  • ESCs embryonic stem cells
  • iPSCs induced pluripotent stem cells
  • hematopoietic progenitor cells include, but are not limited to, bone marrow cells, hematopoietic stem cells (HSCs), embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), and hematopoietic progenitor cells.
  • the base editing system or composition is close to the sequence related to the human HBB mutant gene, and the mutation site (repair of the human HBB gene) is modified by the activity of the base editing enzyme to obtain the wild type HBB gene. .
  • the activity-modifying mutation site (repair of the human HBB gene) using the base-editing enzyme does not alter the amino acid encoded by the HBB gene.
  • the activity modification mutation site (repair of the human HBB gene) using the base editing enzyme increases the expression level of the HBB gene.
  • the obtained repaired human HBB gene is a gene repair produced by introducing a base editing system.
  • the base editing is capable of repairing the HBB:c.-79A>G and HBB:c.-78A>G mutations to wild type A.
  • HBB: c.-79A>G and HBB: c.-78A>G is repaired. effectiveness.
  • a fourth aspect of the invention provides a eukaryotic host cell comprising the base editing system of the first aspect or the composition of the second aspect.
  • a fifth aspect of the invention provides a kit comprising the gRNA sequence or base editing system provided by the first aspect, the non-naturally occurring or engineered composition provided by the second aspect, and the eukaryotic host provided by the fourth aspect One or more of the cells.
  • the kit further comprises a conventional supporting reagent and/or reaction device.
  • the kit can provide one or more reaction or storage buffers.
  • the reagent may be provided in a form usable in a particular assay or in the form of one or more additional components (e.g., in concentrated or lyophilized form) prior to use.
  • the buffer can be any buffer including, but not limited to, sodium carbonate buffer, sodium bicarbonate buffer, borate buffer, Tris buffer, MOPS buffer, HEPES buffer, and combinations thereof.
  • the buffer is basic.
  • the buffer has a pH of from about 7 to about 10.
  • the kit comprises one or more oligonucleotides comprising at least one gRNA sequence as described in the first aspect.
  • kit of the present invention may be provided singly or in combination, and may be provided in any suitable container, such as a vial, bottle, tube or paperboard.
  • a sixth aspect of the invention provides a gRNA sequence or base editing system provided by the first aspect, a non-naturally occurring or engineered composition provided by the second aspect, or a eukaryotic host cell provided by the fourth aspect
  • One or more, fifth A kit for use in the prevention, amelioration, and/or treatment of beta thalassemia.
  • a sixth aspect of the invention provides a gRNA sequence or base editing system as provided by the first aspect, a non-naturally occurring or engineered composition provided by the second aspect, a fourth One or more of the eukaryotic host cells provided by the aspect, a kit provided by the fifth aspect is prevented, improved, and/or treated by HBB: c.-79A>G and HBB: c.-78A> Application of G-mutation-induced beta thalassemia disease.
  • a seventh aspect of the invention provides a gRNA sequence or base editing system as provided by the first aspect, a non-naturally occurring or engineered composition provided by the second aspect, the eukaryotic host cell provided by the fourth aspect Describe one or more methods to apply:
  • the eukaryotic host cell provided in the fourth aspect is administered to a patient for treatment.
  • An eighth aspect of the invention provides a method of treating beta thalassemia disease, comprising:
  • gRNA sequence or base editing system provided by the first aspect, the non-naturally occurring or engineered composition provided by the second aspect, the eukaryotic host cell provided by the fourth aspect, and surgery, biological therapy, immunotherapy One or several combinations of treatments for patients;
  • the gRNA sequence or base editing system provided by the first aspect is provided by the in vitro transfection technique, and the second aspect provides The non-naturally occurring or engineered composition is mixed with the host cell and then the host cell containing the gRNA sequence or base editing system provided by the first aspect, the non-naturally occurring or engineered composition provided by the second aspect is lost Returning to the patient for treatment;
  • the eukaryotic host cell provided in the fourth aspect is introduced into the patient for treatment.
  • FIG. 1 is a schematic diagram showing the positions of HBB gene and HBB: c.-79A>G and HBB:c.-78A>G mutations according to an embodiment of the present invention
  • FIG. 2 is a design diagram of a lentiviral vector according to an embodiment of the present invention.
  • FIG. 3 is an electrophoresis result of PCR identification of a cell line comprising a HBB mutation assay according to an embodiment of the present invention
  • gRNA1-3 corresponds to SEQ ID NO. 53-SEQ ID NO. 55;
  • gRNA1-3 corresponds to SEQ ID NO. 53-SEQ ID NO. 55, respectively.
  • gRNA-1 and BE3 combination can repair 41.7% of the mutation site, and gRNA-2 and BE3 combination can repair 18.2% of the mutation site.
  • the combination of gRNA-2 and BE3 can repair 3.6% of the mutation sites;
  • HF1-BE3 is used herein, and gRNA1-2 corresponds to SEQ ID NO. 53-SEQ ID NO. 54;
  • HF2-BE3 is used herein; gRNA1-2 corresponds to SEQ ID NO. 53-SEQ ID NO. 54;
  • FIG. 9 is a diagram of repairing the #31 293T cell line using the base editing system VRER-HF1-BE3 according to an embodiment of the present invention.
  • FIG. 10 is a Sanger sequencing result of HBB:c.-78A>G of #31#293T cell line repaired by the base editing system VRER-HF1-BE3 according to an embodiment of the present invention; VRER-HF1-BE3 is used here. ; gRNA7-9 corresponds to SEQ ID NO. 66 - SEQ ID NO. 68;
  • FIG. 11 is a Sanger sequencing result of HBB:c.-78A>G of #31#293T cell line repaired by the base editing system VRQR-HF1-BE3 according to an embodiment of the present invention; VRQR-HF1-BE3 is used here. ; gRNA4-6 corresponds to SEQ ID NO. 59 - SEQ ID NO. 61;
  • FIG. 12 is a Sanger sequencing result of HBB:c.-78A>G of #31 No. 293T cell line repaired by the base editing system VRQR-HF1-BE3 according to an embodiment of the present invention; VRQR-HF1-BE3 is used here. ; gRNA7-9 corresponds to SEQ ID NO. 66 - SEQ ID NO. 68;
  • FIG. 13 is a Sanger sequencing result of human skin fibroblasts repairing HBB:c.-78A>G homozygous mutation using base editing system BE3 according to an embodiment of the present invention.
  • BE3 SEQ ID NO. 107
  • gRNA1 corresponds to SEQ ID NO. 53, respectively.
  • FIG. 14 is a Sanger sequencing result of a nuclear transfer embryo repairing HBB:c.-78A>G homozygous mutation using a base editing system BE3 according to an embodiment of the present invention.
  • an embodiment of the present invention provides a base editing system, method, kit, and application thereof for specifically repairing a human HBB gene mutation.
  • the method for specifically repairing a human HBB gene mutation provided by the present invention employs a base editing system or kit for specifically repairing a human HBB gene mutation provided by the present invention, including but not limited to the following steps One or more steps:
  • Primer Premier 5 software was used to design primers to amplify a partial fragment of HBB gene (SEQ ID NO. 106), DNA polymerase was TOYORO KOD FX, and the PCR reaction system and conditions were configured and reacted according to the product specifications.
  • the primer sequence is as follows:
  • the fragment of interest is ligated into the pENTR molecular cloning vector
  • the target fragment and the pENTR/D-TOPO (Thermo Fisher) empty vector were double-digested with Thermo Fast Digest Not1 and Thermo Fast Digest Asc1, and the digestion system was carried out according to a standard molecular cloning method.
  • the purified target fragment was ligated with the linearized pENTR/D-TOPO (Thermo Fisher) empty vector using Thermo T4 DNA ligase (5 U/ ⁇ l), and the ligation system was carried out according to standard molecular cloning methods.
  • the ligation product was transformed into competent cells frozen at -80 ° C, and the Kana-resistant plate was plated and placed in a 37 ° C incubator to allow the competent cells to grow for about 16 hours.
  • Primer Premier 5 software was used to design primers for HBB gene HBB: c.-78A and HBB: c.-79A regions to generate point mutations HBB: c.-78A>G and HBB: c.- 79A>G ( Figure 1 is the implementation of the present invention Examples of HBB gene and HBB: c.-79A>G and HBB: c.-78A>G mutation position map;), DNA polymerase is TOYORO KOD FX, PCR reaction system and conditions are configured according to the instructions and react; primer As follows:
  • NEB Dpn1 was digested to remove the template plasmid, and the restriction enzyme system was carried out according to the standard molecular cloning method.
  • the sequencing primer is M13FP (sequence: TGTAAAACGACGGCCAGT, SEQ ID NO. 109), and obtaining a plasmid containing the mutation of the target fragment (pENTR-HBB (HBB: c.-78A>G) and pENTR-HBB (HBB: c.-79A>G))
  • the HBB fragment was cloned into the pLenti-EF1a-DEST-SFB vector (Invitrogen) to obtain pLenti-HBB (HBB: c.-78A>G) and pLenti-HBB (HBB:c.- 79A>G), the LR reaction system is configured as follows
  • the enzyme was ligated at 25 ° C for 2 hours and then transformed, as described above.
  • FIG. 2 is a schematic diagram of a design of a lentiviral vector according to an embodiment of the present invention.
  • the mutated HBB gene fragment was introduced into 293T cells by a lentiviral vector, and three 293T cell lines including HBB mutations were obtained, which were #2, #18, and #31, respectively.
  • the specific cell line construction steps are as follows:
  • Laying cells Spread the appropriate density of 293T cells in a 6-well plate one day before the transient;
  • Preparation of cells to be infested ensure that the infected cells are in good condition and avoid various contaminations; spread cells in a 6-well plate to control cell density to about 30% at the time of infection, leaving cells with GFP positive control and negative control Hole
  • Recovery culture of infected cells replace the secondary infected virus solution, add fresh medium and resume culture for 24 hours;
  • Monoclonal picking The cells with positive drug screening were manually counted to determine the cell density, and then picked into 96-well plates and picked up according to the amount of 1 cell/100 ⁇ l medium/well. The monoclonal medium was kept at 1 ⁇ g/ml. Drug screening pressure.
  • the monoclonal cells were grown to about 100 cells, the cells were digested with 30 ⁇ l of 0.05% trypsin well, and 200 ⁇ l of fresh medium was added to terminate the digestion reaction. The cells were dispersed in the original wells to uniformly disperse the cells and placed at 37 ° C. The cell culture incubator continues to be cultured.
  • the QIAGEN DNeasy Blood&Tissue Kit (250) kit was used to extract the monoclonal genomic DNA from the 24-well plate.
  • primers were designed to amplify a partial fragment of the HBB gene to detect positive monoclonals; the primers are as follows:
  • FIG. 3 is a result of electrophoresis of PCR-identified cell lines containing HBB mutations according to an embodiment of the present invention.
  • FIG. 4 is a sequencing result of Sanger sequencing to identify a cell line comprising a HBB mutant according to an embodiment of the present invention.
  • the reporter cell line obtained in step 3 is introduced into the base editing (BE) system for targeted repair of the target site.
  • Planting cells The appropriate density of 293T stable cell line (#31 293T cell line) was plated in a 24-well plate one day before the transient;
  • Base-editing enzyme expression vectors include BE3, HF1-BE3, HF2-BE3, VRER-HF1-BE3, VRQR-HF1-BE3. Specifically, we synthesized the amino acids of SEQ ID NO. 95, SEQ ID NO. 97, SEQ ID NO. 99, SEQ ID NO. 103, SEQ ID NO. 105 by conventional gene synthesis and site-directed mutagenesis in the art.
  • the DNA fragment encoding the sequence, and the DNA fragment was ligated into the pcDNA3.1(-) vector (Invitorgen) by digesting the DNA fragment and the vector with XbaI and BamHI to prepare a recombinant vector BE3, HF1- expressing the amino acid sequence.
  • BE3, HF1-BE3 and HF2-BE3 were co-transfected with a gRNA expression vector comprising the sequence of SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, respectively.
  • the gRNA expression vector showing the leader sequence was co-transfected into the cells.
  • the sequencing primer was HBB-BE-FP (sequence: TTCTCAAGCCTCAGACAGTGGT, SEQ ID NO. 116), and the sequencing results were analyzed.
  • the ligation reaction system was configured as follows
  • the sequencing primer was M13-FP (sequence: TGTAAAACGACGGCCAGT, SEQ ID NO. 109). The alignment and analysis of the sequencing results are then performed.
  • gRNA1-3 corresponds to SEQ ID NO. 53-SEQ ID NO. 55, respectively.
  • the arrow indicates the mutation site, and the box indicates the repaired site.
  • BE3 is used.
  • gRNA1-3 corresponds to SEQ ID NO. 53-SEQ ID NO. 55, respectively.
  • gRNA-1 and BE3 combination can repair 41.7% of the mutation site, and gRNA-2 and BE3 combination can repair 18.2% of the mutation site.
  • the combination of gRNA-2 and BE3 repairs 3.6% of the mutation site.
  • HF1-BE3 is used herein, and gRNA1-2 corresponds to SEQ ID NO. 53-SEQ ID NO. 54, respectively.
  • HF2-BE3 is used herein; gRNA1-2 corresponds to SEQ ID NO. 53-SEQ ID NO. 54, respectively.
  • FIG. 9 is a Sanger sequencing result of HBB:c.-78A>G of #31#293T cell line repaired by the base editing system VRER-HF1-BE3 according to an embodiment of the present invention; VRER-HF1-BE3 is used here. ; gRNA4-6 corresponds to SEQ ID NO. 59-SEQ ID NO. 61, respectively.
  • FIG. 10 is a Sanger sequencing result of HBB:c.-78A>G of #31#293T cell line repaired by the base editing system VRER-HF1-BE3 according to an embodiment of the present invention; VRER-HF1-BE3 is used here. ; gRNA7-9 corresponds to SEQ ID NO. 66 - SEQ ID NO. 68, respectively.
  • FIG. 11 is a Sanger sequencing result of HBB:c.-78A>G of #31#293T cell line repaired by the base editing system VRQR-HF1-BE3 according to an embodiment of the present invention; VRQR-HF1-BE3 is used here. ; gRNA4-6 corresponds to SEQ ID NO. 59-SEQ ID NO. 61, respectively.
  • FIG. 12 is a Sanger sequencing result of HBB:c.-78A>G of #31 No. 293T cell line repaired by the base editing system VRQR-HF1-BE3 according to an embodiment of the present invention; VRQR-HF1-BE3 is used here. ; gRNA7-9 corresponds to SEQ ID NO. 66 - SEQ ID NO. 68, respectively.
  • HBB HBB: c.-78A>G
  • HBB human fibroblasts containing HBTB
  • HBB human fibroblasts with HBB (HBB: c.-78A>G) mutations were collected and used P2 Primary Cell Nuclear transfection of the instructions of the X Kit (Lonza);
  • Base-editing enzyme expression vectors include BE3, HF1-BE3, HF2-BE3, VRER-HF1-BE3, VRQR-HF1-BE3. Specifically, we synthesized the amino acids of SEQ ID NO. 95, SEQ ID NO. 97, SEQ ID NO. 99, SEQ ID NO. 103, SEQ ID NO. 105 by conventional gene synthesis and site-directed mutagenesis in the art.
  • the DNA fragment encoding the sequence, and the DNA fragment was ligated into the pcDNA3.1(-) vector (Invitorgen) by digesting the DNA fragment and the vector with XbaI and BamHI to prepare a recombinant vector BE3, HF1- expressing the amino acid sequence.
  • BE3, HF1-BE3 and HF2-BE3 were co-transfected with a gRNA expression vector comprising the sequence of SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, respectively.
  • the gRNA expression vector showing the leader sequence was co-transfected into the cells.
  • P2Primary Cell Instructions for X Kit (Lonza) plus 100 ul of electroporation and transfection of cells according to the instructions;
  • the ligation reaction system was configured as follows
  • the sequencing primer was M13-FP (sequence: TGTAAAACGACGGCCAGT, SEQ ID NO. 116). The alignment and analysis of the sequencing results are then performed.
  • Figure 13 is a Sanger sequencing result of HBB:c.-78A>G homozygous for human skin fibroblasts repaired using base editing system BE3.
  • BE3 SEQ ID NO. 107
  • gRNA1 corresponds to SEQ ID NO. 53, respectively.
  • HBB:c.-78A>G HBB:c.-78A>G
  • HBB human iPS cells with HBB (HBB: c.-78A>G) mutation and use P3Primary Cell Nuclear transfection of the instructions of the X Kit (Lonza);
  • Base-editing enzyme expression vectors include BE3, HF1-BE3, HF2-BE3, VRER-HF1-BE3, VRQR-HF1-BE3. Specifically, we synthesized the amino acids of SEQ ID NO. 95, SEQ ID NO. 97, SEQ ID NO. 99, SEQ ID NO. 103, SEQ ID NO. 105 by conventional gene synthesis and site-directed mutagenesis in the art.
  • the DNA fragment encoding the sequence, and the DNA fragment was ligated into the pcDNA3.1(-) vector (Invitorgen) by digesting the DNA fragment and the vector with XbaI and BamHI to prepare a recombinant vector BE3, HF1- expressing the amino acid sequence.
  • BE3, HF1-BE3 and HF2-BE3 were co-transfected with a gRNA expression vector comprising the sequence of SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, respectively.
  • the gRNA expression vector showing the leader sequence was co-transfected into the cells.
  • P3Primary Cell Instructions for X Kit (Lonza) plus 100 ul of electroporation and transfection of cells according to the instructions;
  • the ligation reaction system was configured as follows
  • HBB human hematopoietic stem cells
  • Hematopoietic stem cells were isolated from peripheral blood of a patient mutated by HBB (HBB: c.-78A>G) and cultured, and a sufficient amount of cells were collected for transfection.
  • HBB HBB: c.-78A>G mutant human hematopoietic stem cells and use P3Primary Cell Nuclear transfection of the instructions of the X Kit (Lonza);
  • Base-editing enzyme expression vectors include BE3, HF1-BE3, HF2-BE3, VRER-HF1-BE3, VRQR-HF1-BE3. Specifically, we synthesized the amino acids of SEQ ID NO. 95, SEQ ID NO. 97, SEQ ID NO. 99, SEQ ID NO. 103, SEQ ID NO. 105 by conventional gene synthesis and site-directed mutagenesis in the art.
  • the DNA fragment encoding the sequence, and the DNA fragment was ligated into the pcDNA3.1(-) vector (Invitorgen) by digesting the DNA fragment and the vector with XbaI and BamHI to prepare a recombinant vector BE3, HF1- expressing the amino acid sequence.
  • BE3, HF1-BE3 and HF2-BE3 were co-transfected with a gRNA expression vector comprising the sequence of SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, respectively.
  • the gRNA expression vector showing the leader sequence was co-transfected into the cells.
  • P3Primary Cell Instructions for X Kit (Lonza) plus 100 ul of electroporation and transfection of cells according to the instructions;
  • the ligation reaction system was configured as follows
  • HBB:c.-78A>G HBB:c.-78A>G mutant human nuclear transfer embryos
  • the eukaryotic expression vector of BE3 is prepared by cloning, the mRNA of BE3 is prepared by transcription or the like, or the protein of BE3 is prepared by prokaryotic and eukaryotic expression.
  • gRNA and BE3 includes but is not limited to the following combinations:
  • the ligation reaction system was configured as follows
  • Table 1 shows the efficiency statistics of a nuclear transfer embryo repairing HBB:c.-78A>G homozygous mutation using the base editing system BE3 according to an embodiment of the present invention.
  • FIG. 14 is a Sanger sequencing result of a nuclear transfer embryo repairing HBB:c.-78A>G homozygous mutation using a base editing system BE3 according to an embodiment of the present invention.

Abstract

提供了碱基编辑系统特异性修复导致人β地中海贫血的A>G致病突变的方法，以及特异性靶向该突变的gRNA及碱基编辑蛋白。该方法可以精确修复人HBB:c-79A>G和HBB:c-78A>G致病突变。将该基因编辑系统导入到人的体细胞或个体中，可以对A>G致病突变进行精确修复，从而治愈β地中海贫血疾病。

Description

用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用
本申请要求了2017年6月15日提交中国专利局的，申请号201710491367.1，发明名称为“用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明涉及基因检测及基因修饰领域，涉及一种用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用。
背景技术
规律成簇间隔短回文重复系统(clustered regularly interspaced short palindromic repeat；CRISPR-associated，CRISPR-Cas)是古菌和细菌的抵抗病毒和质粒侵染的重要免疫防御系统，用来抵抗外源遗传物质的入侵，比如噬菌体病毒和外源质粒。同时，它为细菌提供了获得性免疫：这与哺乳动物的获得性免疫类似，当细菌遭受病毒或者外源质粒入侵时，会产生相应的“记忆”，从而可以抵抗它们的再次入侵。CRISPR/Cas系统可以识别出外源DNA或RNA，并将它们切断，沉默外源基因的表达。正是由于这种精确的靶向功能，CRISPR/Cas系统被开发成一种高效的基因编辑工具。
CRISPR-Cas系统划分为两大类，第一大类CRISPR-Cas系统由多亚基组成的效应复合物发挥功能；第二大类是由单个效应蛋白(如Cas9,Cpf1,C2c1等)来发挥功能。其中，Cas9,Cpf1,C2c1均具有RNA介导的DNA核酸内切酶活性。目前，Cas9和Cpf1蛋白作为基因组编辑工具被广泛应用，克服了传统基因编辑技术步骤繁琐、耗时长、效率低等缺点，以其较少的成分、便捷的操作以及较高的效率满足了大多数领域的基因编辑需求，并有着潜在且巨大的临床应用价值。
在自然界中，CRISPR/Cas系统拥有多种类别，其中CRISPR/Cas9系统是研究最深入，应用最成熟的一种类别。CRISPR-Cas9是一种具有核酸内切酶活性的复合体，识别特定的DNA序列，进行特定位点切割造成双链DNA断裂(Double-strand breaks,DSB)，在没有模板的条件下，发生非同源重组末端连接(Non-homologous end joining,NHEJ)，造成移码突变(frameshift mutation)，导致基因敲除。CRISPR/Cas9是继“锌指核酸内切酶(ZFN)”、“类转录激活因子 效应物核酸酶(TALEN)”之后出现的第三代“基因组定点编辑技术”。凭借着成本低廉，操作方便，效率高等优点，CRISPR/Cas9迅速风靡全球的实验室，成为了生物科研的有力帮手。
CRISPR/Cas9系统是进行基因编辑的强大工具，可以对基因进行定点的精确编辑，它主要包含Cas9和RNA两部分。Cas9靶向切割DNA是通过两种小RNA——crRNA(CRISPR RNA)和tracrRNA(trans-activating crRNA)和靶序列互补识别的原理实现的。现在已经把两种小RNA融合成一条RNA(chimeric RNA)，简称sgRNA(small guide RNA)。crRNA-tracrRNA的二聚体以及sgRNA都能作为向导RNA(guide RNA，gRNA)，介导CRISPR/Cas9系统的寻靶。另外，CRISPR/Cas9介导的DNA定点切割还需要靶位点附近的对各个CRISPR/Cas系统来说都相对保守的PAM(Protospace adjacent motif)序列的辅助，才能识别与gRNA引导序列互补的靶DNA。针对靶位点设计gRNA，Cas9蛋白就会在gRNA的指引下寻找靶DNA,并切割靶DNA。而生物体自身存在着DNA损伤修复的应答机制，会将断裂上下游两端的序列连接起来，从而实现了细胞中目标基因的敲除。如果在此基础上为细胞引入一个修复的模板质粒(供体DNA分子)，这样细胞就会按照提供的模板在修复过程中通过同源重组精确地引入定点突变或者外源片段的定点插入。随着研究的深入，CRISPR/Cas技术已经被广泛的应用。除了基因敲除，基因替换等基础编辑方式，它还可以被用于基因激活，疾病模型构建，甚至是基因治疗。
虽然，CRISPR/Cas9技术的出现，极大地提高了基因定点突变的效率，但目前依然无法满足临床治疗的需要。最近，依托于CRISPR/Cas9技术，科学家开发了新一代的基因编辑技术——碱基编辑技术。碱基编辑系统是由Cytidine deaminase:Cas9:UGI融合蛋白(碱基编辑酶)和gRNA两部分组分组成的精确基因编辑技术5-7。Cytidine deaminase:Cas9:UGI的融合蛋白中的Cas9通过与gRNA结合，并利用gRNA的序列，通过碱基互补配对，将融合蛋白靶向到靶DNA上，然后，碱基编辑酶会利用其中的Cytidine deaminase(胞嘧啶脱氨酶)的酶活性将靶DNAd的4-8位中的C(胞嘧啶)转变成尿嘧啶(Uridine,U)，UGI是尿嘧啶DNA糖基化酶抑制剂(Uracil DNA glycosylase inhibitor,UGI),其通过抑制U的切除，导致DNA在复制时U与A(腺嘌呤,Adenine)配对，然后再通过DNA复制，使得U变成T(Thymine,胸腺嘧啶)，从而最终将C转变成T5,6,8。碱基编辑技术介导的DNA定点突变比通过同源重组技术介导的突变效率高100倍以上，在部分小鼠受精卵中甚至能够达到100％的定点突变效率。根据碱基编辑酶中Cas9是否能够切割靶DNA,将碱基编辑酶命名为Base editor 2(BE2)或者Base editor 3(BE3)，其中BE2完全不能切割双链靶DNA,而BE3能将双链靶DNA中的靶向链(Target strand)切开。
因此，碱基编辑系统能否做到特异性、精确靶向目标基因的靶碱基是碱基编辑系统能否 特异地进行基因的定点突变的先决条件。其中，碱基编辑系统的特异性主要决定于Cas9以及gRNA。通过对Cas9进行定点突变，从而降低Cas9与双链靶DNA的靶向链(Target strand)或非靶向链(Non-target strand)磷酸骨架的非特异性相互作用可以提高特异性。另外，设计、制备并筛选能特异地与靶DNA互补的gRNA也是决定碱基编辑系统效率和特异性的关键因素。
地中海贫血是由红细胞血球蛋白合成异常导致的遗传疾病。患者的红细胞比正常的红细胞更小且更脆弱，非常容易破裂，导致溶血性贫血。另外，其携氧能力也比正常细胞弱，导致全身各个组织缺氧。成年人的血球蛋白包含Hb A2(2.5％)，Hb F(0.5％)和Hb A(97％)三种类型。其中占比最大的HbA蛋白由2个α球蛋白亚基和两个β球蛋白亚基组成1。根据突变亚基的不同，地中海贫血又可以分为α地中海贫血和β地中海贫血。其中β地中海贫血是由血球蛋白β球蛋白亚基突变导致的一种常见单基因遗传疾病，其编码基因为HBB(humanβglobin)1。HBB的突变使得β球蛋白合成减少或者变得不稳定，导致血球蛋白中α球蛋白和β球蛋白的比值失衡，血球蛋白减少，进而影响红细胞的成熟，导致成熟的红细胞数目减少。另外，积累在红细胞中的α球蛋白及其降解产物，会导致红细胞裂解。两种因素叠加在一起，共同导致贫血2。
按照症状的严重程度来分，β地中海贫血可以分为：微型β地中海贫血、中型β地中海贫血和重型β地中海贫血。重型β地中海贫血患者通常在1岁左右出现严重贫血、生长发育停滞、骨骼异常以及肝脾肿大。另外，由于红细胞破裂，会释放血红素中的铁离子，导致血液中铁离子浓度过高，导致肝脏，心脏等多器官的损伤。长期的肝脏、心脏损伤，会导致肝硬化以及心脏衰竭，从而危及生命3。重型β地中海贫血患者如果不接受治疗的话，通常会在5岁之前死亡3。目前，重型β地中海贫血患者需要通过长期输血治疗，或进行骨髓移植治疗。但是长期输血给患者输血，会导致的血铁浓度过高，而辅助治疗的铁离子螯合剂对患者的身体来说也是一种负担。所以，在长期接受输血治疗的患者中，50％的患者也会在35岁之前死亡4。中型β地中海贫血患者的病症比重型的更温和，通常在幼年或晚年出现中度或重度贫血，通常不需要输血治疗。而微型地中海贫血病患者通常不贫血或轻度贫血。
β地中海贫血在地中海区域、非洲、中东、印度次大陆、东南亚以及中国南部非常常见，在有些地区，HBB的突变频率甚至高达10％，在不同地区，HBB的突变位点及其频率有一定的差异2。HBB的突变可以分为两类：第一类为导致β球蛋白无法合成的β0突变，第二类为导致β球蛋白合成量减少的β+突变。HBB:c.-79A>G和HBB:c.-78A>G突变均为β+突变。该突变的杂合子通常不发病，或者只有轻度贫血。而纯合子则会出现重度β地中海贫血病。此外，当人的一条染色体上有HBB:c.-78A>G(或HBB:c.-79A>G)突变，而另外一条染色体 上具有其他类型的β+或β0突变时，也有可能会出现重型地中海贫血。
现在临床上β地中海贫血的治疗方案包括：输血治疗和骨髓移植。长期的输血治疗对患者的身体和心理都是极大的负担，严重地影响了患者的生活质量。而骨髓移植话，需要找到白细胞抗原(Human leukocyte antigen，HLA)配型成功的供体，使得其应用范围受到一定的限制。传统的基因治疗通过提取患者的造血干细胞(Hematopoietic stem cell,HSC)或骨髓细胞，并通逆转录病毒或者慢病毒载体将β球蛋白或γ球蛋白编码序列导入HSC，从而提高患者的血球蛋白的水平，从而治疗重度地中海贫血，但是传统基因治疗具有外源基因表达量差异大、效率低等缺点，导致不同患者治疗效果差异大，部分人能完全摆脱输血治疗，而另外一部分人则仍然需要输血治疗。此外，传统基因治疗还可能因为病毒的随机整合，导致基因突变，从而引发致癌等安全隐患。而提取患者自身的造血干细胞(Hematopoietic stem cell,HSC)或骨髓细胞，利用碱基编辑系统修复HBB:c.-79A>G和HBB:c.-78A>G突变，则能在原基因座上精确修复突变位点，具有效率高、安全性高等特点。将突变修复了的HSC回输给患者，则能治愈患者的重度β地中海贫血。
目前，碱基编辑系统已经应用于植物、酵母、人细胞和小鼠胚胎的单碱基基因编辑8-10，但碱基编辑系统是否能够用于修复HBB:c.-79A>G和HBB:c.-78A>G突变，并在细胞水平上治疗由以上突变导致的β地中海贫血病仍然未知。
因此，有必要提供一种用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用。
在本文中提及或通过引用结合在本文中的任何文献中的任何产品的任何制造商的说明书、说明、产品规格、和产品表，通过引用并入本文，并且可以在本发明的实践中采用。更具体地说，所有参考的文献均通过引用并入本文，其程度如同每个单独的文献被确切地并单独地指明通过引用而并入本文。
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发明内容
为解决上述问题，本发明提供了一种用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用。
本发明第一方面提供了一种用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，包含碱基编辑酶和gRNA，所述碱基编辑酶为融合蛋白，所述融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、胞嘧啶脱氨酶(Cytidine deaminase)结构域以及尿嘧啶DNA糖基化酶抑制(Uracil DNA glycosylase inhibitor，UGI)结构域，所述gRNA包括但不限于化脓性链球菌Cas9(Streptococcus pyogenes Cas9,SpCas9)的Sp-gRNA，金黄色葡萄球菌Cas9(Staphylococcus aureus Cas9,SaCas9)的Sa-gRNA,空肠弯曲杆菌Cas9(Campylobacter jejuni Cas9,CjCas9)的Cj-gRNA,嗜热链球菌Cas9(Streptococcus thermophilus Cas9,StCas9)的St-gRNA,脑膜炎双球菌Cas9(Neisseria meningitidis Cas9,NmCas9)的Nm-gRNA,毛螺菌科Cpf1(Lachnospiraceae Cpf1,LbCpf1)的Lb-gRNA,氨基酸球菌Cpf1(Acidaminococcus Cpf1,AsCpf1)的As-gRNA中的一种或多种。
优选地，所述gRNA包含约15-100个核苷酸，并且还包括与靶DNA序列互补的至少12个连续核苷酸组成的引导序列。
优选地，所述gRNA与靶DNA互补的序列包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种。
具体地，所述的与gRNA引导序列互补的靶DNA序列为邻近HBB:c.-79A>G和HBB:c.-78A>G突变的人基因组DNA序列。
优选地，所述的gRNA为如下1)或2)中的一种：
1)包括crRNA和tracrRNA，其中，crRNA的部分序列与tracrRNA的部分序列互补、并组成二聚体，其中，crRNA的引导序列包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种；
2)嵌合型单链sgRNA，其中sgRNA由crRNA与tracrRNA融合而成，sgRNA的引导序列的包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种。
本领域技术人员可以理解的是，gRNA除包含与靶DNA序列互补的引导序列外，还包含骨架序列。gRNA的形式可以是由crRNA和tracrRNA组成的二聚体，也可以是人工改造的由crRNA和tracrRNA融合而成的嵌合型单链sgRNA。
本领域技术人员可以理解的是，CRISPR RNA(crRNA)的RNA分子利用它的一部分序列(3’端)与被称作tracrRNA(trans-activating crRNA)的RNA分子通过碱基配对结合在一起，形成嵌合RNA(即tracrRNA/crRNA二聚体)，然后，借助crRNA的另一部分序列(5’端)与靶DNA位点进行碱基配对，以这种方式，这种嵌合RNA就能够引导所述的碱基编辑酶结合到靶DNA序列上并进行剪辑编辑。
本领域技术人员可以理解的是，在实际应用时，还可以将tracrRNA和crRNA融合在一起形成单向导RNA(single guide RNA,sgRNA)，即本发明所述的嵌合型单链sgRNA，所述的嵌合型单链sgRNA能够引导所述的碱基编辑酶结合到靶DNA序列上并进行剪辑编辑。
本发明实施方式中，所述CRISPR/Cas系统的效应蛋白结构域为无切割活性或仅具有单链切割活性的CRISPR/Cas系统的效应蛋白。本发明一实施方式中，所述CRISPR/Cas系统的效应蛋白包括但不限定于化脓性链球菌Cas9(Streptococcus pyogenes Cas9,SpCas9)，金黄色葡萄球菌Cas9(Staphylococcus aureus Cas9,SaCas9),空肠弯曲杆菌Cas9(Campylobacter jejuni Cas9,CjCas9),嗜热链球菌Cas9(Streptococcus thermophilus Cas9,StCas9),脑膜炎双球菌Cas9(Neisseria meningitidis Cas9,NmCas9),毛螺菌科Cpf1(Lachnospiraceae Cpf1,LbCpf1)，氨基酸球菌Cpf1(Acidaminococcus Cpf1,AsCpf1)等效应蛋白的重组或突变体。可以理解的是，这些效应蛋白的重组或突变体为无切割活性或仅具有单链切割活性的Cas蛋白。
本发明一实施方式中，所述CRISPR/Cas系统的效应蛋白结构域包括但不限定于国际申请号为：PCT/US2016/058345或CT/US2016/058344国际公开版本中公开的重组Cas9蛋白。
具体地，PCT/US2016/058345国际公开版本权利要求1、2公开的重组Cas9蛋白结构域的氨基酸序列中，如一实施方式中，满足如下条件：
“与PCT/US2016/058345国际公开版本中公开的SEQ ID NO.9-262所示氨基酸序列至少80％、85％、90％、92％、95％、96％、97％、98％、99％或99.5％一致，且与天然存在的Cas9蛋白不完全一致；
其中，SEQ ID NO.9-262中的任一条序列至少在262、267、294、405、409、480、543、694、1219、1224、1256、1362氨基酸位点中含有一个、两个、三个、四个、五个、六个或七个突变”。
本发明一实施方式中，所述碱基编辑酶包括但不限定于国际申请号为：PCT/US2016/058345或CT/US2016/058344国际公开版本中公开的碱基编辑融合蛋白(即本发明第一方面所述的碱基编辑酶)。
现有文献公开的碱基编辑融合蛋白(即本发明第一方面所述的碱基编辑酶)同样适用本发明实施例，所述现有文献包括但不限于：Genome-wide target specificities of CRISPR RNA-guided programmable deaminase，Nature Biotechnology，2017April 10。
本发明一实施方式中，所述的胞嘧啶脱氨酶结构域包括但不限定于大鼠的APOBEC1，小鼠的APOBEC3，人类的APOBEC1，人类的APOBEC3A，人类的APOBEC3B，人类的APOBEC3C，人类的APOBEC3D，人类的APOBEC3F，人类的APOBEC3G，人类的APOBEC3H,人类的AID，七鳃鳗的CDA1等胞嘧啶脱氨酶。
本发明一实施方式中，所述的尿嘧啶DNA糖基化酶抑制结构域包括但不限定于枯草杆菌噬菌体PBS1的尿嘧啶DNA糖基化酶抑制剂(UGI)以及枯草杆菌噬菌体PBS2的尿嘧啶DNA糖基化酶抑制剂(UGI)。
可以理解的是，PCT/US2016/058345或PCT/US2016/058344国际公开版本中公开的、适用于碱基编辑的重组Cas9蛋白结构域、胞嘧啶脱氨酶结构域、UGI结构域或由“重组Cas9蛋白结构域、胞嘧啶脱氨酶结构域、UGI结构域”构成的融合蛋白(即本发明第一方面所述的碱基编辑酶)，本领域技术人员在不脱离本发明实施例原理的前提下，可以做出合适的选择。
本发明一优选实施方式中，所述碱基编辑酶的氨基酸序列包含但不限于与SEQ ID NO.94-105所示氨基酸序列至少80％、85％、90％、92％、95％、96％、97％、98％、99％或99.5％一致的序列。
具体地，所述SEQ ID NO.94-105所示氨基酸序列分别对应BE2,BE3,HF1-BE2,HF1-BE3,HF2-BE2,HF2-BE3,VQR-HF1-BE2,VQR-HF1-BE3,VRER-HF1-BE2,VRER-HF1-BE3,VRQR-HF1-BE2,VRQR-HF1-BE3的编码氨基酸序列。
具体地，我们通过本领域常规的基因合成和定点突变的方式合成了以上氨基酸序列的编码核苷酸序列。具体地，合成所述SEQ ID NO.94-105的氨基酸序列的编码DNA序列，并通过用XbaⅠ和BamHⅠ的酶切和连接克隆到pcDNA3.1(-)载体(Invitorgen)中，制得表达所述氨基酸序列的重组载体。
本发明一实施方式中，由引导序列和骨架序列组成的gRNA，可采用将gRNA对应的编 码DNA序列克隆到包含T7或Sp6启动子的载体中(如pDR274,Addgene)，或通过PCR、退火、合成等方式直接在gRNA对应的编码DNA的前端加上T7或Sp6启动子，经转录可获得转录产物gRNA序列。
本发明一实施例中，可以直接合成gRNA，其中gRNA可以是普通的gRNA，也可以是经过化学修饰的gRNA，化学修饰包括对RNA骨架以及碱基的修饰。
本发明一实施例中，gRNA可通过完全互补或基本互补、或以一定互补百分比，特异性地识别所述HBB突变基因(本发明实施例中，“HBB突变基因”与“靶核苷酸序列”可以互换)。
本发明所提供的gRNA与所述靶核苷酸序列具有足够互补性以便与该靶核苷酸序列杂交并且指导碱基编辑酶与该靶核苷酸序列的特异性结合。在一些实施例中，gRNA与其相应的靶核苷酸序列之间的互补程度是约或多于约50％、60％、75％、80％、85％、90％、95％、97.5％、99％或更多。
本发明实施例中，“互补”是指核酸与另一个核酸序列借助于传统的沃森-克里克碱基配对或其他非传统类型形成一个或多个氢键。“互补百分比”表示一个核酸分子中可与一个第二核酸序列形成氢键(例如，沃森-克里克碱基配对)的残基的百分比(例如，10个之中有5、6、7、8、9、10个即为50％、60％、70％、80％、90％、和100％互补)。“完全互补”表示一个核酸序列的所有连续残基与一个第二核酸序列中的相同数目的连续残基形成氢键。如本文使用的“基本上互补”是指在一个具有8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、30、35、40、45、50个或更多个核苷酸的区域上至少为60％、65％、70％、75％、80％、85％、90％、95％、97％、98％、99％、或100％的互补程度。
本发明一实施方式中，所述gRNA是引导序列与骨架序列组成的成熟的gRNA序列。
本发明一实施方式中，所述引导序列为表1中SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一条或多条。
本发明实施方式中，所述gRNA骨架序列为本领域技术人员常规使用的，该骨架序列可以是由crRNA和tracrRNA(trans-activating crRNA)组成的二聚体，也可以是人工改造的由crRNA和tracrRNA融合而成的嵌合型单链sgRNA。gRNA的骨架序列可以是目前常规使用的序列，如GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC，也可以是在此基础上构建的一些突变体，这些突变体与目前常规使用的序列有50％、60％、75％、80％、85％、90％、95％、97.5％、99％或更高的相似度。
本发明实施方式中，所述gRNA序列可特异性识别的靶核苷酸可以包括与HBB疾病相关 基因或多核苷酸。
“疾病相关”基因或多核苷酸是指与非疾病对照的组织或细胞相比，在来源于疾病影响的组织的细胞中以异常水平或以异常形式产生转录或翻译产物的任何基因或多核苷酸。在改变的表达与疾病的出现和/或进展相关的情况下，它可以是一个以异常高的水平被表达的基因；它可以是一个以异常低的水平被表达的基因。疾病相关基因还指具有一个或多个突变或直接负责或与一个或多个负责疾病的病因学的基因连锁不平衡的遗传变异的基因。转录的或翻译的产物可以是已知的或未知的，并且可以处于正常或异常水平。
具体地，所述特异性修复的人HBB基因突变包括但不限于人HBB:c.-79A>G和HBB:c.-78A>G突变。
在本发明实施方式中，术语“靶核苷酸”是指核糖核苷酸或其类似物。“靶核苷酸”可以包含一个或多个经修饰的核苷酸，如甲基化的核苷酸和核苷酸类似物。
本发明一实施方式中，所述的碱基编辑酶融合蛋白中，CRISPR/Cas系统的效应蛋白结构域与胞嘧啶脱氨酶(Cytidine deaminase)结构域之间还连接有第一连接肽。
本发明一实施方式中，所述的碱基编辑酶融合蛋白中，所述CRISPR/Cas系统的效应蛋白结构域与尿嘧啶DNA糖基化酶抑制(Uracil DNA glycosylase inhibitor，UGI)结构域之间还连接有第二连接肽。
本发明一实施方式中，所述第一连接肽为(GGGS)n、(GGGGS)n、(G)n、(EAAAK)n、(GGS)n、(SGGS)n、SGSETPGTSESATPES或XP)n中的一种，其中n为1-30的自然数，X为任一一种氨基酸。
本发明一实施方式中，所述第一连接肽为XTEN连接多肽。
本发明一实施方式中，所述第二连接肽为(GGGGS)n、(G)n、(EAAAK)n、(GGS)n、(SGGS)n、SGSETPGTSESATPES、SGGS或XP)n中的一种，其中n为1-30的自然数，X为任一一种氨基酸。
本发明一实施方式中，所述第二连接肽为XTEN连接多肽。
本发明一实施方式中，所述第一连接肽和第二连接肽可以相同或不同。
在本发明一实施方式中，所述的碱基编辑酶融合蛋白中还包括其他任选地在任何两个结构域之间的连接肽段，所述连接肽段具体地，包括但不限于表位标签、报告基因序列、以及具有下列活性的一者或多者的蛋白质结构域：甲基酶活性、脱甲基酶活性、转录激活活性、转录阻遏活性、转录释放因子活性、组蛋白修饰活性、RNA切割活性和核酸结合活性。表位标签的非限制性实例包括组氨酸(His)标签、V5标签、FLAG标签、流感病毒血凝素(HA)标签、Myc标签、VSV-G标签、和硫氧还蛋白(Trx)标签。报告基因的实例包括，但不限于， 谷胱甘肽-S-转移酶(GST)、辣根过氧化物酶(HRP)、氯霉素乙酰转移酶(CAT)、β-半乳糖苷酶、β-葡糖醛酸糖苷酶、萤光素酶、绿色荧光蛋白(GFP)、HcRed、DsRed、青荧光蛋白(CFP)、黄色荧光蛋白(YFP)、以包括蓝色荧光蛋白(BFP)的自发荧光蛋白。
在本发明一实施方式中，所述的碱基编辑酶融合蛋白中还可以融合一种蛋白质或蛋白质片段，所述蛋白质或蛋白质片段结合DNA分子或结合其他细胞分子，其包括，但不限于，麦芽糖结合蛋白(MBP)、S-tag、Lex A DNA结合结构域(DBD)融合物、GAL4DNA结合结构域融合物、以及单纯疱疹病毒(HSV)BP16蛋白融合物。在本发明一些实施例中，所述融合蛋白质为分子标记，使用标记的碱基编辑酶融合蛋白可用来鉴定靶序列的位置。
术语“多肽”、“肽”和“蛋白质”在本文可互换地使用，是指具有任何长度的氨基酸的聚合物。该聚合物可以是可以是直链或支链的，它可以包含修饰的氨基酸，并且它可以被非氨基酸中断。这些术语还涵盖已经被修饰的氨基酸聚合物；这些修饰例如二硫键形成、糖基化、脂化(lipidation)、乙酰化、磷酸化、或任何其他修饰，如与检测分子标记组分的结合。
本发明第二方面提供了一种非天然存在的或工程化的组合物，所述组合物为如下1)-6)中的一种或多种：
1)包含一种或多种载体，该一种或多种载体包含组分I和组分II：
所述组分I包括第一调节元件，以及与所述第一调节元件可操作地连接的编码如第一方面所述的碱基编辑酶的编码序列；所述组分II包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如第一方面所述的gRNA的编码序列；其中，组分I和II位于相同或不同载体上；
2)包含如第一方面所述的碱基编辑酶的mRNA以及载体，所述载体包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如第一方面所述的gRNA的编码序列；
3)包含如第一方面所述的碱基编辑酶的蛋白以及载体，所述载体包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如第一方面所述的gRNA的编码序列；
4)包含如第一方面所述的碱基编辑酶的表达载体以及如第一方面所述的gRNA；
5)包含如第一方面所述的碱基编辑酶的mRNA以及如第一方面所述的gRNA；
6)包含如第一方面所述的碱基编辑酶的蛋白以及如第一方面所述的gRNA。
可以理解的是，在本发明实施方式中，第一方面所述的碱基编辑酶可以是DNA,RNA或蛋白的形式；gRNA可以是DNA或RNA的形式。
在本发明实施方式中，术语“载体”是指一种核酸分子，它能够运送与其连接的另一种核酸分子。载体包括但不限于，单链、双链、或部分双链的核酸分子；包括一个或多个自由端、无自由端(例如环状的)的核酸分子；包括DNA、RNA、或两者的核酸分子；以及本领域 已知的其他多种多样的多核苷酸。可选地，一种类型的载体是“质粒”，其是指其中可以例如通过标准分子克隆技术插入另外的DNA片段的环状双链DNA环。可选地，另一种类型的载体是病毒载体，其中病毒衍生的DNA或RNA序列存在于用于包装病毒(例如，逆转录病毒、复制缺陷型逆转录病毒、腺病毒、复制缺陷型腺病毒、以及腺相关病毒)的载体中。病毒载体还包含由用于转染到一种宿主细胞中的病毒携带的多核苷酸。某些载体(例如，具有细菌复制起点的细菌载体和附加型哺乳动物载体)能够在它们被导入的宿主细胞中自主复制。其他载体(例如，非附加型哺乳动物载体)在引入宿主细胞后整合到该宿主细胞的基因组中，并且由此与该宿主基因组一起复制。而且，某些载体能够指导它们可操作连接的基因的表达。这样的载体在此被称为“表达载体”。在重组DNA技术中使用的普通表达栽体通常是质粒形式。
通常，在载体内，“可操作地连接”旨在表示核苷酸序列以一种允许该核苷酸序列的表达的方式被连接至一个或多个调节元件(可选地，载体处于一种体外转录/翻译系统中可以表达该核苷酸序列；可选地，当该载体被引入到宿主细胞中时可以表达该核苷酸序列)。
在本发明实施方式中，术语“表达”是指从DNA模板转录成多核苷酸(如转录成mRNA或其他RNA转录物)的过程和/或转录的mRNA随后翻译成肽、多肽或蛋白质的过程。转录物和编码的多肽可以总称为“基因产物”。如果多核苷酸来源于基因组DNA，表达可以包括真核细胞中mRNA的剪接。
本文使用的术语“非天然存在的”或“工程化的”可互换地使用，当指核酸分子或多肽时，表示该核酸分子或多肽至少基本上从它们在自然界中或如发现于自然界中的与其结合的至少另一种组分游离出来。
在本发明优选实施方式中，所述的第一调节元件(在本发明一实施例中，本发明所述的“调节元件”可以理解为“基因表达盒”)包括一个或多个pol III启动子(例如1、2、3、4、5、或更多个pol III启动子)、一个或多个pol II启动子(例如1、2、3、4、5、或更多个pol II启动子)、一个或多个pol I启动子(例如1、2、3、4、5、或更多个pol I启动子)、或其组合。此外，第一调节元件还可以是T7启动子，Sp6启动子等。pol III启动子的实例包括但不限于U6和H1启动子。pol II启动子的实例包括但不限于逆转录劳斯肉瘤病毒(RSV)LTR启动子(任选地具有RSV增强子)、巨细胞病毒(CMV)启动子(任选地具有CMV增强子)[参见，例如，波沙特(Boshart)等人，《细胞》(Cell)41:521-530(1985)]、SV40启动子、二氢叶酸还原酶启动子、β-肌动蛋白启动子、磷酸甘油激酶(PGK)启动子、和EF1α启动子。
在本发明一些实施方式中，蛋白编码序列经密码子优化，以便在特定的细胞如真核细胞中表达。这些真核细胞可以是特定生物的那些或来源于特定生物，如哺乳动物，包括但不限于人、小鼠、大鼠、兔、狗、或非人类灵长动物。一般而言，密码子优化是指通过用在宿主 细胞的基因中更频繁地或者最频繁地使用的密码子代替天然序列的至少一个密码子(例如约或多于约1、2、3、4、5、10、15、20、25、50个、或更多个密码子同时维持该天然氨基酸序列而修饰一个核酸序列以便增强在感兴趣宿主细胞中的表达的方法。不同的物种对于具有特定氨基酸的某些密码子展示出特定的偏好。密码子偏好性(在生物之间的密码子使用的差异)经常与信使RNA(mRNA)的翻译效率相关，而该翻译效率则被认为依赖于(除其他之外)被翻译的密码子的性质和特定的转运RNA(tRNA)分子的可用性。细胞内选定的tRNA的优势一般反映了最频繁用于肽合成的密码子。因此，可以将基因定制为基于密码子优化在给定生物中的最佳基因表达。密码子利用率表可以容易地获得，例如在密码子使用数据库(“Codon Usage Database”)中，并且这些表可以通过不同的方式调整适用。参见，中村Y.(Nakamura Y.)等人，“从国际DNA序列数据库中制表的密码子使用：2000年的状态”(Codon usage tabulated from the international DNA sequence databases:status for the year 2000)《核酸研究》(Nucl.Acids Res.)28：292(2000年)。用于密码子优化特定的数列以便在特定的宿主细胞中表达的计算机算法也是可得的，如基因制造(Gene Forge)(Aptagen公司；雅各布斯(Jacobus)，PA)，也是可得的。在一些实施例中，在编码CRISPR酶的序列中的一个或多个密码子(例如1、2、3、4、5、10、15、20、25、50个、或更多个、或所有密码子)相应于对于特定氨基酸最频繁使用的密码子。
在本发明优选实施方式中，所述的第二调节元件包括但不限定于一个或多个pol II启动子(例如1、2、3、4、5、或更多个pol II启动子)、一个或多个pol I启动子(例如1、2、3、4、5、或更多个pol I启动子)、或其组合,pol III启动子(包括但不限定于U6,H1启动子),T7启动子，Sp6启动子等。
在本发明优选实施方式中，所述的第一或二调节元件还包括增强子、内部核糖体进入位点(IRES)、和其他表达控制元件(例如转录终止信号，如多聚腺苷酸化信号和多聚U序列)。这样的调节序列例如描述于戈德尔(Goeddel)，《基因表达技术：酶学方法》(GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY)185，学术出版社(Academic Press)，圣地亚哥(San Diego)，加利福尼亚州(1990)中。调节元件包括指导一个核苷酸序列在许多类型的宿主细胞中的组成型表达的那些序列以及指导该核苷酸序列只在某些宿主细胞中表达的那些序列(例如，组织特异型调节序列)。组织特异型启动子可主要指导在感兴趣的期望组织中的表达，所述组织例如肌肉、神经元、骨、皮肤、血液、特定的器官(例如肝脏、胰腺)、或特殊的细胞类型(例如淋巴细胞)。调节元件还可以时序依赖性方式(如以细胞周期依赖性或发育阶段依赖性方式)指导表达，该方式可以是或者可以不是组织或细胞类型特异性的。
本发明第三方面提供了一种用于在体细胞或人体内特异性修复人HBB基因突变的方法，所述方法包括递送第一方面所述的碱基编辑系统或第二方面所述的组合物，使所述的碱基编辑系统或组合物与人HBB突变基因相关的序列接近，获得经过修复的人HBB基因。
在本发明实施方式中，术语“使所述的碱基编辑系统或组合物与人HBB突变基因相关的序列接近”是指将组分递送到体内离体(in vitro)或(in vivo)环境中，离体环境如本领域技术人员根据具体实验需求配置的反应溶液,体内环境比如细胞内；所述的术语“接近”是指在离体(in vitro)或体内(in vivo)环境中，各组分可以与人HBB突变基因相关的序列接触，并在一定的条件下发生本领域技术人员可以预料的反应。可以理解的是，在本发明实施方式中，术语“人体内”可以是离体(in vitro)或体内(in vivo)，比如，可以是人生殖系内；值得注意的是，在本发明同日申请的另一篇专利中，本申请人特别要求保护一种用于在人生殖系内特异性修复人HBB基因突变的方法，特别的，另一篇专利中所述人生殖系包括人生殖细胞、受精卵或胚胎；在本发明第三方面所述的、对应的本发明权利要求保护范围中，“人体内”不包括在本发明同日申请的另一篇专利对应的权利要求请求保护的“生殖系内”)。
在本发明一实施方式中，本发明提供了以下方法，包括向体细胞或人体内递送一个或多个多核苷酸、一个或多个载体、一个或多个转录本、和/或一个或多个转录的蛋白。在一些方面，本发明进一步提供了通过这样的方法产生的细胞，受精卵及胚胎。
在本发明一实施方式中，将与gRNA组合的碱基编辑系统复合物递送至体细胞或人体内。可以使用常规的病毒和非病毒基的基因转移方法将第一方面所述的碱基编辑系统或第二方面所述的组合物引入人的体细胞或个体中。
可以使用这样的方法向细胞培养物或个体中的细胞给予第二方面所述的组合物。非病毒载体递送系统包括DNA质粒、RNA(例如在此描述的载体的转录本)、蛋白、裸核酸以及与递送赋形剂(如脂质体)复合的核酸。病毒载体递送系统包括DNA和RNA病毒，在被递送至细胞后它们具有游离型或整合型基因组。关于基因递送系统的综述，参见安德(Anderson)，《科学》(Science)256:808-813(1992)；纳贝尔(Nabel)&费尔格纳(Felgner)，TIBTECH11:211-217(1993)；三谷(Mitani)&卡斯基(Caskey)，TIBTECH 11:162-166(1993)；狄龙(Dillon)，TIBTECH 11:167-175(1993)；米勒(Miller)，《自然》(Nature)357:455-460(1992)；范·布朗特(Van Brunt)，《生物技术》(Biotechnology)6(10):1149-1154(1988)；维涅(Vigne)，《恢复神经学和神经科学》(Restorative Neurology and Neurosciece)8:35-36(1995)；克雷默(Kremer)&佩里科德特(Perricaudet)，《英国医学公报》(British Medical Bulletin)51(1):31-44(1995)；哈嗒嗒(Haddada)等人，在《微生物学和免疫学当前主题》(Current Topics in Microbiologyand Immunology)中多尔夫勒(Doerfler)和博姆(编辑)(1995)； 以及余(Yu)等人，《基因疗法》(Gene Therapy)1:13-26(1994)。
核酸的非病毒递送方法包括脂转染、核转染、显微注射、电转、基因枪、病毒颗粒、脂质体、免疫脂质体、聚阳离子或脂质:核酸共轭物、裸DNA、人工病毒体以及DNA的试剂增强性摄取。脂转染描述于例如美国专利号5,049,386、4,946,787和4,897,355中并且脂转染试剂是市售的(例如，TransfectamTM和LipofectinTM)。适于多核苷酸的有效的受体识别脂转染的阳离子和中性脂质包括Felgner(费尔格纳)，WO 91/17424；WO 91/16024的那些。递送可以针对细胞(例如体外或离体给予)或靶组织(例如体内给予)。
脂质:核酸复合物(包括靶向的脂质体，如免疫脂质复合物)的制备是本领域的技术人员熟知的(参见例如，克丽丝特尔(Crystal)，《科学》(Science)270:404-410(1995)；布莱泽(Blaese)等人，《癌症基因疗法》(Cancer Gene Ther.)2:291-297(1995)；贝尔(Behr)等人，《生物共轭化学》(Bioconjugate Chem.)5:382-389(1994)；雷米(Remy)等人，《生物共轭化学》5:647-654(1994)；高(Gao)等人，《基因疗法》(Gene Therapy)2:710-722(1995)；艾哈迈德(Ahmad)等人，《癌症研究》(Cancer Res.)52:4817-4820(1992)；美国专利号4,186,183、4,217,344、4,235,871、4,261,975、4,485,054、4,501,728、4,774,085、4,837,028以及4,946,787)。
在本发明一优选实施方式中，先制备第一方面所述的碱基编辑酶的真核表达载体、原核表达载体或者体外转录载体；然后将真核表达载体扩增，并制备无内毒素的转染级的真核表达载体(或将原核表达载体转入到大肠杆菌中诱导表达碱基编辑酶)，制备碱基编辑酶的蛋白。还可以将碱基编辑酶的编码DNA克隆到包含T7或者Sp6启动子的体外转录载体中，通过体外转录，制备碱基编辑酶的mRNA。
在本发明一优选实施方式中，制备第一方面所述的gRNA,可以直接合成gRNA,或者将gRNA的编码序列克隆到包含T7或Sp6启动子的转录载体(如pDR274，来自Addgene)，或通过PCR、退火、合成等方式直接在gRNA对应的编码DNA的前端加上T7或Sp6启动子，再通过体外转录的方法制备gRNA；或者将所述的gRNA的编码序列克隆到真核表达载体pGEM-T-U6-gRNA载体中，所述的pGEM-T-U6-gRNA载体为通过PCR的方式从pX330载体(Addgene)上扩增U6启动子以及gRNA的骨架序列，将PCR产物通过TA克隆的方式连入到pGEM-T载体(Promega)中所制得。
在本发明一优选实施方式中，将第一方面所述的碱基编辑酶的DNA(转录mRNA或者翻译的蛋白)与gRNA的表达载体(或者转录的gRNA，或者合成的gRNA)通过包括但不限于脂质体转染、病毒感染、电转和显微注射等方式导入到人的体细胞或人体中。
进一步地，所述病毒感染采用的病毒载体包括但不限定于逆转录病毒载体、慢病毒载体、 腺病毒载体和腺病毒先关病毒载体等。
在本发明一优选实施方式中，人细胞包括但不限定于骨髓细胞，造血干细胞(HSC)，胚胎干细胞(ESC)，诱导多能性干细胞(iPSC)和造血祖细胞。
可以理解的是，所述的碱基编辑系统或组合物与人HBB突变基因相关的序列接近，利用碱基编辑酶的活性修饰突变位点(修复人HBB基因的突变)，获得野生型HBB基因。
在本发明一具体实施方式中，所述的利用碱基编辑酶的活性修饰突变位点(修复人HBB基因的突变)并不改变HBB基因编码的氨基酸。
在本发明一具体实施方式中，所述的利用碱基编辑酶的活性修饰突变位点(修复人HBB基因的突变)提高了HBB基因的表达水平。
在本发明一优选实施方式中，所述获得经过修复的人HBB基因是通过引入碱基编辑系统而产生的基因修复。
进一步地，所述碱基编辑能够将HBB:c.-79A>G和HBB:c.-78A>G突变修复成野生型的A。
在本发明一优选实施方式中，包括但不限于采用Sanger测序、TA克隆测序或者深度测序等方式检测目标细胞或组织HBB:c.-79A>G和HBB:c.-78A>G被修复的效率。
本发明第四方面提供了一种真核宿主细胞，包含第一方面所述的碱基编辑系统或第二方面所述的组合物。
本发明第五方面提供了一种试剂盒，包含第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞中的一种或多种。
本发明一实施方式中，所述试剂盒还包括常规配套的反应试剂和/或反应设备。例如，试剂盒可以提供一种或多种反应或存储缓冲液。可以按在具体测定中可用的形式或按在使用之前需要添加一种或多种其他组分的形式(例如按浓缩或冻干形式)提供试剂。缓冲液可以是任何缓冲液，包括但不限于碳酸钠缓冲液、碳酸氢钠缓冲液、硼酸盐缓冲液、Tris缓冲液、MOPS缓冲液、HEPES缓冲液及其组合。在一些实施例中，该缓冲液是碱性的。在一些实施例中，该缓冲液具有从约7至约10的pH。在一些实施例中，该试剂盒包括一个或多个寡核苷酸，该一种或多种核酸包含至少一种如第一方面所述的gRNA序列。
本发明所述试剂盒中的各组分可以单独地或组合地提供，并且可以被提供于任何适合的容器中，如小瓶、瓶子、管或纸板。
本发明第六方面提供了一种如第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞中的一种或多种、第五 方面提供的一种试剂盒在预防、改善、和/或治疗β地中海贫血病中的应用。
在本发明一优选实施方式中，本发明第六方面提供了一种如第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞中的一种或多种、第五方面提供的一种试剂盒在预防、改善、和/或治疗由HBB:c.-79A>G和HBB:c.-78A>G突变导致的β地中海贫血疾病中的应用。
本发明第七方面提供了一种如第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞按下述一种或多种方法进行应用：
1)将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞单独进行应用；
2)将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞与手术、生物治疗、免疫治疗中的一种或几种联合应用；
3)采用体内投递的方式将所述第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞直接投递到患者体内进行治疗；
4)先通过体外转染技术将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物与宿主细胞混合，然后将含有第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物的宿主细胞输回患者体内实施治疗；
5)将第四方面提供的真核宿主细胞输入患者体内实施治。
本发明第八方面提供了一种β地中海贫血疾病的治疗方法，包括：
1)将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞单独对患者进行治疗；
2)将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞与手术、生物治疗、免疫治疗中的一种或几种联合对患者进行治疗；
3)采用体内投递的方式将所述第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物、第四方面提供的真核宿主细胞直接投递到患者体内进行治疗；
4)先通过体外转染技术将第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的 非天然存在的或工程化的组合物与宿主细胞混合，然后将含有第一方面提供的gRNA序列或碱基编辑系统、第二方面提供的非天然存在的或工程化的组合物的宿主细胞输回患者体内实施治疗；
5)将第四方面提供的真核宿主细胞输入患者体内实施治疗。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本申请的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例提供的HBB基因及HBB:c.-79A>G和HBB:c.-78A>G突变位置示意图；
图2为本发明实施例提供的慢病毒载体设计图；
图3为本发明实施例提供的PCR鉴定包含HBB突变测细胞系的电泳结果；
图4为本发明实施例提供的Sanger测序鉴定包含HBB突变细胞系的测序结果；
图5为本发明实施例提供的利用碱基编辑系统BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为BE3；gRNA1-3分别对应SEQ ID NO.53-SEQ ID NO.55；
图6为本发明实施例提供的利用碱基编辑系统BE3修复#31号293T细胞系的HBB:c.-78A>G的TA克隆测序结果；
箭头标注的为突变位点，方框标注的为修复后的位点，此处使用的为BE3；
gRNA1-3分别对应SEQ ID NO.53-SEQ ID NO.55，经过统计，gRNA-1和BE3组合能够修复41.7％的突变位点，gRNA-2和BE3组合能修复18.2％的突变位点，而gRNA-2和BE3的组合能修复3.6％的突变位点；
图7为本发明实施例提供的利用碱基编辑系统HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为HF1-BE3，gRNA1-2分别对应SEQ ID NO.53-SEQ ID NO.54；
图8为本发明实施例提供的利用碱基编辑系统HF2-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为HF2-BE3；gRNA1-2分别对应SEQ ID NO.53-SEQ ID NO.54；
图9为本发明实施例提供的利用碱基编辑系统VRER-HF1-BE3修复#31号293T细胞系 的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRER-HF1-BE3；gRNA4-6分别对应SEQ ID NO.59-SEQ ID NO.61；
图10为本发明实施例提供的利用碱基编辑系统VRER-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRER-HF1-BE3；gRNA7-9分别对应SEQ ID NO.66-SEQ ID NO.68；
图11为本发明实施例提供的利用碱基编辑系统VRQR-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRQR-HF1-BE3；gRNA4-6分别对应SEQ ID NO.59-SEQ ID NO.61；
图12为本发明实施例提供的利用碱基编辑系统VRQR-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRQR-HF1-BE3；gRNA7-9分别对应SEQ ID NO.66-SEQ ID NO.68；
图13为本发明实施例提供的利用碱基编辑系统BE3修复HBB:c.-78A>G纯合突变的人皮肤成纤维细胞Sanger测序结果。此处使用的为BE3(SEQ ID NO.107)。gRNA1分别对应SEQ ID NO.53。
图14为本发明实施例提供的利用碱基编辑系统BE3修复HBB:c.-78A>G纯合突变的核移植胚胎的Sanger测序结果。
具体实施方式
以下所述是本发明实施例的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明实施例原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也视为本发明实施例的保护范围。
若无特别说明，本发明实施例中所用试剂及耗材均为市售商品。
除非另有说明，本发明的实践采用免疫学、生物化学、化学、分子生物学、微生物学、细胞生物学、基因组学和重组DNA的常规技术，这些在本领域的技能之内。参见萨姆布鲁克(Sambrook)、弗里奇(Fritsch)和马尼亚蒂斯(Maniatis)，《分子克隆：实验室手册》(MOLECULAR CLONING:A LABORATORY MANUAL)，第2次编辑(1989)；《当代分子生物学实验手册》(CURRENT PROTOCOLS IN MOLECULAR BIOLOGY)(F.M.奥苏贝尔(F.M.Ausubel)等人编辑，(1987))；《酶学方法》(METHODS IN ENZYMOLOGY)系列(学术出版公司)：《PCR2:实用方法》(PCR 2:A PRACTICAL APPROACH)(M.J.麦克弗森(M.J.MacPherson)、B.D.黑姆斯(B.D.Hames)和G.R.泰勒(G.R.Taylor)编辑(1995))、哈洛(Harlow)和拉内(Lane)编辑(1988)《抗体：实验室手册》(ANTIBODIES,A LABORATORY  MANUAL)，以及《动物细胞培养》(ANIMAL CELL CULTURE)(R.I.弗雷谢尼(R.I.Freshney)编辑(1987))。
本发明一具体实施方式中，本发明实施例提供了一种用于特异性修复人HBB基因突变的碱基编辑系统、方法、试剂盒及其应用。
本发明提供的用于特异性修复人HBB基因突变的方法，采用了本发明提供的用于特异性修复人HBB基因突变的碱基编辑系统或试剂盒，所述方法包括但不限于如下步骤的一个或多个步骤：
一.HBB基因片段的克隆
1.目的片段的扩增
以HEK293T细胞的基因组为模板，利用Primer Premier 5软件设计引物扩增HBB基因的部分片段(SEQ ID NO.106)，DNA聚合酶为TOYORO KOD FX，PCR反应体系及条件按照产品说明书配置并进行反应；引物序列如下所示:
Figure PCTCN2017098360-appb-000001
1)PCR扩增产物的纯化(QIAGEN PCR Purification Kit)。
2.目的片段连接到pENTR分子克隆载体中
1)利用Thermo FastDigest Not1和Thermo FastDigest Asc1对目的片段和pENTR/D-TOPO(Thermo Fisher)空载体进行双酶切，酶切体系按照标准分子克隆方法进行。
2)双酶切产物的纯化(QIAGEN PCR Purification Kit)。
3)利用Thermo T4 DNA ligase(5U/μl)连接纯化后的目的片段与线性化的pENTR/D-TOPO(Thermo Fisher)空载体，连接体系按照标准分子克隆方法进行。
4)连接产物转化到冻存于-80℃的感受态细胞中，涂卡纳抗性的平板，置于37℃恒温培养箱使感受态生长约16小时。
5)挑取单个克隆测序，测序引物为M13-FP(序列为：TGTAAAACGACGGCCAGT，SEQ ID NO.109)，获得含有目的片段的质粒(pENTR-HBB)。
二.pLenti-EF1a-HBB(HBB:c.-78A>G)和pLenti-EF1a-HBB(HBB:c.-79A>G)慢病毒质粒的构建
1)以pENTR-HBB为模板，利用Primer Premier 5软件针对HBB基因HBB:c.-78A和HBB:c.-79A区域设计引物产生点突变HBB:c.-78A>G和HBB:c.-79A>G(图1为本发明实施 例提供的HBB基因及HBB:c.-79A>G和HBB:c.-78A>G突变位置示意图；)，DNA聚合酶为TOYORO KOD FX，PCR反应体系及条件按照说明书配置并进行反应；引物如下所示：
HBB-78-QC-FP:
Figure PCTCN2017098360-appb-000002
HBB-78-QC-RP:
Figure PCTCN2017098360-appb-000003
HBB-79-QC-FP:
Figure PCTCN2017098360-appb-000004
HBB-79-QC-RP:
Figure PCTCN2017098360-appb-000005
2)PCR扩增产物的纯化(QIAGEN PCR Purification Kit)。
3)NEB Dpn1酶切除去模板质粒，酶切体系按照标准分子克隆方法进行。
4)酶切产物的纯化(QIAGEN PCR Purification Kit)。
5)酶切产物的转化，方法同上。
6)挑取单个克隆测序，测序引物为M13FP(序列为：TGTAAAACGACGGCCAGT，SEQ ID NO.109)，获得含有目的片段突变的质粒(pENTR-HBB(HBB:c.-78A>G)和pENTR-HBB(HBB:c.-79A>G))
7)利用Gataway分子克隆方法，将HBB片段克隆到pLenti-EF1a-DEST-SFB载体(Invitrogen)，从而获得pLenti-HBB(HBB:c.-78A>G)和pLenti-HBB(HBB:c.-79A>G)，配置LR反应体系如下
Figure PCTCN2017098360-appb-000006
LR反应体系混匀后，于25℃酶连仪连接2小时后进行转化，方法同上。
8)挑取单个克隆测序，测序引物为EF1a-FP，获得含有目的片段突变的质粒(pLenti-HBB(HBB:c.-78A>G)或pLenti-HBB(HBB:c.-79A>G))
图2为本发明实施例提供的慢病毒载体设计图。本发明实施例通过慢病毒载体将突变的HBB基因片段导入到293T细胞中，获得3个包含HBB突变的293T细胞系，分别为#2、#18、#31。具体的细胞系构建步骤如下步骤三：
三.慢病毒介导的293T稳转报告细胞系的构建
稳转细胞系构建步骤：
第1天：
铺细胞：于瞬转前一天铺适当密度的293T细胞于6孔板中；
第2天：
瞬转：6孔板中按pLenti-HBB(HBB:c.-78A>G)质粒或pLenti-HBB(HBB:c.-79A>G)质粒(以pLenti-eGFP质粒为阳性对照):psPAX2:pMD2.G＝4:3:1(1.2μg:0.9μg:0.3μg)的比例，使用1xPEI瞬转试剂(质粒(μg):1xPEI(μl)＝1:3)；瞬转8小时后换液；瞬转24小时后观察GFP荧光，确定瞬转效率；
第3天：
待侵染细胞的准备：确保被侵染细胞的状态良好，避免各种污染；铺细胞于6孔板中，控制细胞密度至侵染时约30％，留出GFP阳性对照和阴性对照的细胞用孔；
第4天：
病毒液收集与侵染：收集瞬转48小时后的293T细胞上清(原293T细胞中重新加入2ml新鲜培养基继续培养)，用0.45um滤头过滤病毒液；按照病毒液体积：新鲜培养基＝2:1的比例加入新鲜培养基后混匀；1:800加入polybrene(stock solution:8mg/ml)，混匀后加入到相应的待侵染细胞中；于预热的37℃离心机中离心侵染(600g X 90min，升降加速度均设为1)；离心结束后取出细胞，于37℃细胞培养箱中继续培养侵染8小时后换新鲜培养基。
第5天：
病毒的二次侵染：二次病毒侵染之前，先观察被侵染细胞的GFP荧光，以确定第一次病毒侵染的效果；取瞬转72小时的293T病毒液重复上述步骤，进行离心侵染。
第6天：
被侵染细胞的恢复培养：换掉二次侵染的病毒液，加入新鲜培养基恢复培养24小时；
第7天：
细胞的筛选：根据pLenti-HBB(HBB:c.-78A>G)和pLenti-EF1a-HBB(HBB:c.-79A>G)质粒载体上的真核抗性基因采用Puromycin进行药物筛选)；药物筛选浓度为1μg/ml，采用悬浮筛选的方法(用0.05％的胰酶于37℃消化恢复培养后的293T细胞，铺35％密度的细胞到新的6孔板中进行筛选；悬浮药筛24小时后换液去除漂浮的死细胞，加药继续药筛，直至阴性对照组细胞全部死亡，本次实验共筛选3天。
第8天：
单克隆的挑取：将药物筛选阳性的细胞手动计数确定细胞密度后挑到96孔板中，按照1个细胞/100μl培养基/孔的量挑取，单克隆培养基中继续保持1μg/ml的药物筛选压力。
第9-15天：
挑单克隆后一周内可不用观察，待其生长；
第16-22天：
开始观察单克隆，每隔一天观察一次，只挑取一个克隆/孔的细胞，做好标记；
第23天:
待单克隆细胞生长约100个细胞左右时，用30μl 0.05％的胰酶原孔消化细胞后，加入200μl新鲜培养基终止消化反应，原孔中吹散细胞，使细胞均匀分散，置于37℃细胞培养箱继续培养。
第26天：
待单克隆细胞长满96孔时，重复胰酶消化步骤，每孔传2/3细胞至24孔板中用于单克隆检测，其余1/3细胞传至新的24孔板中用于后续实验。
第30天：
用QIAGEN DNeasy Blood&Tissue Kit(250)试剂盒提取24孔板中单克隆基因组DNA，以此为模板，设计引物扩增HBB基因的部分片段，以检测阳性单克隆；引物如下所示:
Figure PCTCN2017098360-appb-000007
图3为本发明实施例提供的PCR鉴定包含HBB突变测细胞系的电泳结果。
图4为本发明实施例提供的Sanger测序鉴定包含HBB突变细胞系的测序结果。
四.在步骤三中获得的报告细胞系导入碱基编辑(BE)体系用于靶位点的定向修复
1.细胞培养与转染
1)铺细胞：于瞬转前一天铺适当密度的293T稳转细胞系(#31号293T细胞系)于24孔板中；
2)瞬转：24孔板中按照碱基编辑酶表达载体:gRNA表达载体＝2:1(1μg:0.5μg)的比例进行，同时设置单独瞬转碱基编辑酶表达载体和Lenti-eGFP的组作为阴性对照。碱基编辑酶表达载体包括BE3,HF1-BE3,HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。具体地，我们通过本领域常规的基因合成和定点突变的方式合成所述SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.103、SEQ ID NO.105的氨基酸序列的编码DNA片段，并通过用XbaⅠ和BamHⅠ酶切DNA片段和载体，将DNA连接克隆到pcDNA3.1(-)载体(Invitorgen)中，制得表达所述氨基酸序列的重组载体BE3、HF1-BE3、HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。
其中，BE3,HF1-BE3和HF2-BE3分别与包含SEQ ID NO.53，SEQ ID NO.54，SEQ ID NO.55引导序列的gRNA表达载体共转染细胞。VRER-HF1-BE3,VRQR-HF1-BE3分别与含SEQ ID NO.59,SEQ ID NO.60,SEQ ID NO.61,SEQ ID NO.66,SEQ ID NO.67,SEQ ID NO.68所示引导序列的gRNA表达载体共转染细胞。
使用1xPEI瞬转试剂(质粒(μg):1xPEI(μl)＝1:3)；瞬转8小时后换液；瞬转24小时后观察GFP荧光，确定瞬转效率；
2.基因突变的修复效率检测
1)瞬转48小时后收集细胞，用QIAGEN DNeasy Blood&Tissue Kit(250)试剂盒提取细胞基因组DNA，以此为模板，用检测单克隆的引物扩增目的片段；
2)目的片段的回收与测序，测序引物为HBB-BE-FP(序列为：TTCTCAAGCCTCAGACAGTGGT，SEQ ID NO.116)，并进行测序结果分析。
3)TA克隆检测BE靶位点编辑效率
a.将目的片段的PCR产物连接到T载体(Promega)中
按照标准分子克隆实验方法，配置连接反应体系如下
Figure PCTCN2017098360-appb-000008
连接反应体系混匀后，于22℃酶连仪连接1小时
b.转化，方法同上。
于涂平板前一小时准备蓝白斑筛选用板：按照无抗培养基:IPTG(0.5M):X-gal(20mg/ml)＝40μl:10μl:50μl的比例避光混匀各种试剂，均匀涂在对应抗性的平板上，避光倒置于37℃细菌培养箱中待用；
c.待细菌生长约16小时后，挑白色菌斑克隆送测，测序引物为M13-FP(序列为：TGTAAAACGACGGCCAGT,SEQ ID NO.109)。然后进行测序结果的比对与分析。
图5为本发明实施例提供的利用碱基编辑系统BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为BE3；gRNA1-3分别对应SEQ ID NO.53-SEQ ID NO.55。
图6为本发明实施例提供的利用碱基编辑系统BE3修复#31号293T细胞系的HBB:c.-78A>G的TA克隆测序结果。
箭头标注的为突变位点，方框标注的为修复后的位点，此处使用的为BE3。
gRNA1-3分别对应SEQ ID NO.53-SEQ ID NO.55，经过统计，gRNA-1和BE3组合能够修复41.7％的突变位点，gRNA-2和BE3组合能修复18.2％的突变位点，而gRNA-2和BE3的组合能修复3.6％的突变位点。
图7为本发明实施例提供的利用碱基编辑系统HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为HF1-BE3，gRNA1-2分别对应SEQ ID NO.53-SEQ ID NO.54。
图8为本发明实施例提供的利用碱基编辑系统HF2-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；
此处使用的为HF2-BE3；gRNA1-2分别对应SEQ ID NO.53-SEQ ID NO.54。
图9为本发明实施例提供的利用碱基编辑系统VRER-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRER-HF1-BE3；gRNA4-6分别对应SEQ ID NO.59-SEQ ID NO.61。
图10为本发明实施例提供的利用碱基编辑系统VRER-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRER-HF1-BE3；gRNA7-9分别对应SEQ ID NO.66-SEQ ID NO.68。
图11为本发明实施例提供的利用碱基编辑系统VRQR-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRQR-HF1-BE3；gRNA4-6分别对应SEQ ID NO.59-SEQ ID NO.61。
图12为本发明实施例提供的利用碱基编辑系统VRQR-HF1-BE3修复#31号293T细胞系的HBB:c.-78A>G的Sanger测序结果；此处使用的为VRQR-HF1-BE3；gRNA7-9分别对应SEQ ID NO.66-SEQ ID NO.68。
五.在HBB(HBB:c.-78A>G)突变的病人人成纤维细胞中导入碱基编辑(BE)体系用于靶位点的定向修复
1.细胞培养与转染
1)从HBB(HBB:c.-78A>G)突变的病人手臂内侧分裂一块直径3mm的皮肤，然后将皮肤组织至于细胞培养皿中进行培养，等到皮肤细胞从皮肤组织中爬到细胞培养皿中，并且细胞密度达到80％左右时，将细胞传代，获得大量病人来源的包含HBB(HBB:c.-78A>G)突变的人成纤维细胞。
2)转染当天，收集HBB(HBB:c.-78A>G)突变的人成纤维细胞，并用P2 Primary Cell
Figure PCTCN2017098360-appb-000009
X Kit(Lonza)的说明书进行核转染；
3)瞬转：24孔板中按照碱基编辑酶表达载体:gRNA表达载体＝2:1(1μg:0.5μg)的比例进行，同时设置单独瞬转碱基编辑酶表达载体和Lenti-eGFP的组作为阴性对照。碱基编辑酶表达载体包括BE3,HF1-BE3,HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。具体地，我们通过本领域常规的基因合成和定点突变的方式合成所述SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.103、SEQ ID NO.105的氨基酸序列的编码DNA片段，并通过用XbaⅠ和BamHⅠ酶切DNA片段和载体，将DNA连接克隆到pcDNA3.1(-)载体(Invitorgen)中，制得表达所述氨基酸序列的重组载体BE3、HF1-BE3、HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。
其中，BE3,HF1-BE3和HF2-BE3分别与包含SEQ ID NO.53，SEQ ID NO.54，SEQ ID NO.55引导序列的gRNA表达载体共转染细胞。VRER-HF1-BE3,VRQR-HF1-BE3分别与含SEQ ID NO.59,SEQ ID NO.60,SEQ ID NO.61,SEQ ID NO.66,SEQ ID NO.67,SEQ ID NO.68所示引导序列的gRNA表达载体共转染细胞。按照P2Primary Cell
Figure PCTCN2017098360-appb-000010
X Kit(Lonza)的说明书加100ul的电转液，并按照说明书的程序转染细胞；
2.基因突变的修复效率检测
1)转染48小时后收集细胞，用QIAGEN DNeasy Blood&Tissue Kit(250)试剂盒提取细胞基因组DNA，以此为模板，用检测如下引物扩增目的片段；
HBB-T7E1-FP
Figure PCTCN2017098360-appb-000011
HBB-T7E1-RP
Figure PCTCN2017098360-appb-000012
2)目的片段的回收与测序，测序引物为HBB-T7E1-FP，并进行测序结果分析。
3)TA克隆检测BE靶位点编辑效率
a.将目的片段的PCR产物连接到T载体(Promega)中
按照标准分子克隆实验方法，配置连接反应体系如下
Figure PCTCN2017098360-appb-000013
连接反应体系混匀后，于22℃酶连仪连接1小时
b.转化，方法同上。
于涂平板前一小时准备蓝白斑筛选用板：按照无抗培养基:IPTG(0.5M):X-gal(20mg/ml)＝40μl:10μl:50μl的比例避光混匀各种试剂，均匀涂在对应抗性的平板上，避光倒置于37℃细菌培养箱中待用；
c.待细菌生长约16小时后，挑白色菌斑克隆送测，测序引物为M13-FP(序列为：TGTAAAACGACGGCCAGT,SEQ ID NO.116)。然后进行测序结果的比对与分析。
图13为利用碱基编辑系统BE3修复人皮肤成纤维细胞纯合的HBB:c.-78A>G的Sanger测序结果。此处使用的为BE3(SEQ ID NO.107)。gRNA1分别对应SEQ ID NO.53。
六.在HBB(HBB:c.-78A>G)突变的人iPS细胞中导入碱基编辑(BE)体系用于靶位点的定向修复
1.细胞培养与转染
1)从HBB(HBB:c.-78A>G)突变的病人的皮肤成纤维细胞诱导产生包含HBB(HBB:c.-78A>G)突变iPS细胞。
2)转染当天，收集HBB(HBB:c.-78A>G)突变的人iPS细胞，并用P3Primary Cell
Figure PCTCN2017098360-appb-000014
X Kit(Lonza)的说明书进行核转染；
3)瞬转：24孔板中按照碱基编辑酶表达载体:gRNA表达载体＝2:1(1μg:0.5μg)的比例进行，同时设置单独瞬转碱基编辑酶表达载体和Lenti-eGFP的组作为阴性对照。碱基编辑酶表达载体包括BE3,HF1-BE3,HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。具体地，我们通过本领域常规的基因合成和定点突变的方式合成所述SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.103、SEQ ID NO.105的氨基酸序列的编码DNA片段，并通过用XbaⅠ和BamHⅠ酶切DNA片段和载体，将DNA连接克隆到pcDNA3.1(-)载体(Invitorgen)中，制得表达所述氨基酸序列的重组载体BE3、HF1-BE3、HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。
其中，BE3,HF1-BE3和HF2-BE3分别与包含SEQ ID NO.53，SEQ ID NO.54，SEQ ID NO.55引导序列的gRNA表达载体共转染细胞。VRER-HF1-BE3,VRQR-HF1-BE3分别与含SEQ ID NO.59,SEQ ID NO.60,SEQ ID NO.61,SEQ ID NO.66,SEQ ID NO.67,SEQ ID NO.68所示引导序列的gRNA表达载体共转染细胞。按照P3Primary Cell
Figure PCTCN2017098360-appb-000015
X Kit(Lonza)的说明书加100ul的电转液，并按照说明书的程序转染细胞；
2.基因突变的修复效率检测
1)转染48小时后收集细胞，用QIAGEN DNeasy Blood&Tissue Kit(250)试剂盒提取细胞 基因组DNA，以此为模板，用检测如下引物扩增目的片段；
HBB-T7E1-FP
Figure PCTCN2017098360-appb-000016
HBB-T7E1-RP
Figure PCTCN2017098360-appb-000017
2)目的片段的回收与测序，测序引物为HBB-T7E1-FP，并进行测序结果分析。
3)TA克隆检测BE靶位点编辑效率
a.将目的片段的PCR产物连接到T载体(Promega)中
按照标准分子克隆实验方法，配置连接反应体系如下
Figure PCTCN2017098360-appb-000018
连接反应体系混匀后，于22℃酶连仪连接1小时
b.转化，方法同上。
于涂平板前一小时准备蓝白斑筛选用板：按照无抗培养基:IPTG(0.5M):X-gal(20mg/ml)＝40μl:10μl:50μl的比例避光混匀各种试剂，均匀涂在对应抗性的平板上，避光倒置于37℃细菌培养箱中待用；
c.待细菌生长约16小时后，挑白色菌斑克隆送测，测序引物为M13-FP。然后进行测序结果的比对与分析。
七.在HBB(HBB:c.-78A>G)突变的人造血干细胞(HSC)中导入碱基编辑(BE)体系用于靶位点的定向修复
1.细胞培养与转染
1)从HBB(HBB:c.-78A>G)突变的病人的外周血中分离造血干细胞并培养，收集足够量的细胞用于转染。
2)转染当天，收集HBB(HBB:c.-78A>G)突变的人造血干细胞，并用P3Primary Cell
Figure PCTCN2017098360-appb-000019
X Kit(Lonza)的说明书进行核转染；
3)瞬转：24孔板中按照碱基编辑酶表达载体:gRNA表达载体＝2:1(1μg:0.5μg)的比例进行，同时设置单独瞬转碱基编辑酶表达载体和Lenti-eGFP的组作为阴性对照。碱基编辑酶表达载体包括BE3,HF1-BE3,HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。具体地，我们通过本领域常规的基因合成和定点突变的方式合成所述SEQ ID NO.95、SEQ ID NO.97、SEQ ID NO.99、SEQ ID NO.103、SEQ ID NO.105的氨基酸序列的编码DNA片段，并通过用XbaⅠ和BamHⅠ酶切DNA片段和载体，将DNA连接克隆到pcDNA3.1(-)载体(Invitorgen)中，制得表达所述氨基酸序列的重组载体BE3、HF1-BE3、HF2-BE3,VRER-HF1-BE3,VRQR-HF1-BE3。
其中，BE3,HF1-BE3和HF2-BE3分别与包含SEQ ID NO.53，SEQ ID NO.54，SEQ ID NO.55引导序列的gRNA表达载体共转染细胞。VRER-HF1-BE3,VRQR-HF1-BE3分别与含SEQ ID NO.59,SEQ ID NO.60,SEQ ID NO.61,SEQ ID NO.66,SEQ ID NO.67,SEQ ID NO.68所示引导序列的gRNA表达载体共转染细胞。按照P3Primary Cell
Figure PCTCN2017098360-appb-000020
X Kit(Lonza)的说明书加100ul的电转液，并按照说明书的程序转染细胞；
2.基因突变的修复效率检测
1)转染48小时后收集细胞，用QIAGEN DNeasy Blood&Tissue Kit(250)试剂盒提取细胞基因组DNA，以此为模板，用检测如下引物扩增目的片段；
HBB-T7E1-FP
Figure PCTCN2017098360-appb-000021
HBB-T7E1-RP
Figure PCTCN2017098360-appb-000022
2)目的片段的回收与测序，测序引物为HBB-T7E1-FP，并进行测序结果分析。
3)TA克隆检测BE靶位点编辑效率
a.将目的片段的PCR产物连接到T载体(Promega)中
按照标准分子克隆实验方法，配置连接反应体系如下
Figure PCTCN2017098360-appb-000023
连接反应体系混匀后，于22℃酶连仪连接1小时
b.转化，方法同上。
于涂平板前一小时准备蓝白斑筛选用板：按照无抗培养基:IPTG(0.5M):X-gal(20mg/ml)＝40μl:10μl:50μl的比例避光混匀各种试剂，均匀涂在对应抗性的平板上，避光倒置于37℃细菌培养箱中待用；
c.待细菌生长约16小时后，挑白色菌斑克隆送测，测序引物为M13-FP。然后进行测序结果的比对与分析。
八.在HBB(HBB:c.-78A>G)突变的人核移植胚胎的中导入碱基编辑(BE)体系用于靶位点的定向修复
1)通过克隆的方式制备靶向HBB(HBB:c.-78A>G)突变的gRNA表达载体，通过合成或者转录等方式制备靶向HBB(HBB:c.-78A>G)突变的gRNA的RNA。
2)通过克隆的方式制备BE3的真核表达载体，通过转录等相关方式制备BE3的mRNA，或者通过原核和真核表达的方式制备BE3的蛋白。
3)通过显微注射或电转的方式将gRNA(100ng/ul)和BE3(200ng/ul)导入到人核移植的HBB(HBB:c.-78A>G)纯合突变的胚胎中，gRNA和BE3包含但不限定于以下几种组合：
a.gRNA表达载体和BE3真核表达载体
b.gRNA表达载体和BE3mRNA
c.gRNA表达载体和BE3蛋白
d.gRNA的RNA和BE3真核表达载体
e.gRNA的RNA和BE3mRNA
f.gRNA的RNA和BE3蛋白
2.基因突变的修复效率检测
1)导入48小时后收集细胞，用收集胚胎提取DNA，以此为模板，用HBB-T7E1-FP和
HBB-T7E1-RP引物扩增目的片段；
HBB-T7E1-FP
Figure PCTCN2017098360-appb-000024
HBB-T7E1-RP
Figure PCTCN2017098360-appb-000025
2)目的片段的回收与测序，测序引物为HBB-T7E1-FP，并进行测序结果分析。
3)TA克隆检测BE靶位点编辑效率
a.将目的片段的PCR产物连接到T载体(Promega)中
按照标准分子克隆实验方法，配置连接反应体系如下
Figure PCTCN2017098360-appb-000026
连接反应体系混匀后，于22℃酶连仪连接1小时
b.转化，方法同上。
于涂平板前一小时准备蓝白斑筛选用板：按照无抗培养基:IPTG(0.5M):X-gal(20mg/ml)＝40μl:10μl:50μl的比例避光混匀各种试剂，均匀涂在对应抗性的平板上，避光倒置于37℃细菌培养箱中待用；
c.待细菌生长约16小时后，挑白色菌斑克隆送测，测序引物为M13-FP。然后进行测序结果的比对与分析。
表1为本发明实施例提供的利用碱基编辑系统BE3修复HBB:c.-78A>G纯合突变的核移植胚胎的效率统计。
表1.
Figure PCTCN2017098360-appb-000027
图14为本发明实施例提供的利用碱基编辑系统BE3修复HBB:c.-78A>G纯合突变的核移植胚胎的Sanger测序结果。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。
Figure PCTCN2017098360-appb-000028
Figure PCTCN2017098360-appb-000029
Figure PCTCN2017098360-appb-000030
Figure PCTCN2017098360-appb-000031
Figure PCTCN2017098360-appb-000032
Figure PCTCN2017098360-appb-000033
Figure PCTCN2017098360-appb-000034
Figure PCTCN2017098360-appb-000035
Figure PCTCN2017098360-appb-000036
Figure PCTCN2017098360-appb-000037
Figure PCTCN2017098360-appb-000038
Figure PCTCN2017098360-appb-000039
Figure PCTCN2017098360-appb-000040
Figure PCTCN2017098360-appb-000041
Figure PCTCN2017098360-appb-000042
Figure PCTCN2017098360-appb-000043
Figure PCTCN2017098360-appb-000044
Figure PCTCN2017098360-appb-000045
Figure PCTCN2017098360-appb-000046
Figure PCTCN2017098360-appb-000047
Figure PCTCN2017098360-appb-000048
Figure PCTCN2017098360-appb-000049
Figure PCTCN2017098360-appb-000050
Figure PCTCN2017098360-appb-000051
Figure PCTCN2017098360-appb-000052
Figure PCTCN2017098360-appb-000053
Figure PCTCN2017098360-appb-000054
Figure PCTCN2017098360-appb-000055
Figure PCTCN2017098360-appb-000056
Figure PCTCN2017098360-appb-000057
Figure PCTCN2017098360-appb-000058
Figure PCTCN2017098360-appb-000059
Figure PCTCN2017098360-appb-000060
Figure PCTCN2017098360-appb-000061
Figure PCTCN2017098360-appb-000062
Figure PCTCN2017098360-appb-000063
Figure PCTCN2017098360-appb-000064
Figure PCTCN2017098360-appb-000065
Figure PCTCN2017098360-appb-000066
Figure PCTCN2017098360-appb-000067
Figure PCTCN2017098360-appb-000068
Figure PCTCN2017098360-appb-000069
Figure PCTCN2017098360-appb-000070
Figure PCTCN2017098360-appb-000071
Figure PCTCN2017098360-appb-000072
Figure PCTCN2017098360-appb-000073

Claims (13)

  1. 一种用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，包含碱基编辑酶和gRNA，所述碱基编辑酶为融合蛋白，所述融合蛋白包括CRISPR/Cas系统的效应蛋白结构域、胞嘧啶脱氨酶结构域以及尿嘧啶DNA糖基化酶抑制剂结构域。
  2. 如权利要求1所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述gRNA包含15-100个核苷酸，并且还包括与靶DNA序列互补的至少12个连续核苷酸组成的引导序列。
  3. 如权利要求1或2所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述的gRNA的引导序列包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种。
  4. 如权利要求1或2所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述的gRNA为如下1)或2)中的一种：
    1)包括crRNA和tracrRNA，其中，crRNA的部分序列与tracrRNA的部分序列互补、并组成二聚体，其中，crRNA的引导序列包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种；
    2)嵌合型单链sgRNA，其中sgRNA由crRNA与tracrRNA融合而成，sgRNA的引导序列的包括SEQ ID NO.1-SEQ ID NO.93所示核苷酸序列中的一种或多种。
  5. 如权利要求1所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述碱基编辑酶的氨基酸序列包括与SEQ ID NO.94-105所示氨基酸序列至少80％、85％、90％、92％、95％、96％、97％、98％、99％或99.5％一致的序列。
  6. 如权利要求1所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述的碱基编辑酶融合蛋白中，CRISPR/Cas系统的效应蛋白结构域与胞嘧啶脱氨酶结构域之间还连接有第一连接肽。
  7. 如权利要求1所述用于特异性修复人HBB基因突变的碱基编辑系统，其特征在于，所述的碱基编辑酶融合蛋白中，所述CRISPR/Cas系统的效应蛋白结构域与尿嘧啶DNA糖基化酶抑制结构域之间还连接有第二连接肽。
  8. 一种非天然存在的或工程化的组合物，其特征在于，所述组合物为1)-6)中的一种或多种：
    1)包含一种或多种载体，该一种或多种载体包含组分I和组分II：
    所述组分I包括第一调节元件，以及与所述第一调节元件可操作地连接的编码如权利要求1所述的碱基编辑酶的编码序列；所述组分II包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如权利要求1所述的gRNA的编码序列；其中，组分I和II位于相同或不同载体上；
    2)包含如权利要求1所述的碱基编辑酶的mRNA以及载体，所述载体包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如权利要求1所述的gRNA的编码序列；
    3)包含如权利要求1所述的碱基编辑酶的蛋白以及载体，所述载体包括第二调节元件，以及与所述第二调节元件可操作地连接的编码如权利要求1所述的gRNA的编码序列；
    4)包含如权利要求1所述的碱基编辑酶的表达载体以及如权利要求1所述的gRNA；
    5)包含如权利要求1所述的碱基编辑酶的mRNA以及如权利要求1所述的gRNA；
    6)包含如权利要求1所述的碱基编辑酶的蛋白以及如权利要求1所述的gRNA。
  9. 一种用于在体细胞或个体内特异性修复人HBB基因突变的方法，其特征在于，包括：递送如权利要求1所述的碱基编辑系统或如权利要求8所述的组合物，使所述的碱基编辑系统或组合物与人HBB突变基因相关的序列接近，获得经过修复的人HBB基因。
  10. 一种真核宿主细胞，其特征在于，包含如权利要求1所述的碱基编辑系统或如权利要求8所述的组合物。
  11. 一种用于在体细胞或人体内特异性修复人HBB基因突变的试剂盒，其特征在于，包括权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物、权利要求10提供的真核宿主细胞中的一种或多种。
  12. 一种如权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存 在的或工程化的组合物、权利要求10提供的真核宿主细胞、权利要求11提供的试剂盒在体细胞或人体内预防、改善、和/或治疗由HBB:c.-79A>G和HBB:c.-78A>G突变导致的β地中海贫血疾病中的应用。
  13. 一种如权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物、权利要求10提供的真核宿主细胞按下述一种或多种方法进行应用：
    1)将权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物、权利要求10提供的真核宿主细胞单独进行应用；
    2)将权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物、权利要求10提供的真核宿主细胞与手术、生物治疗、免疫治疗中的一种或几种联合应用；
    3)采用体内投递的方式将所述如权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物、权利要求10提供的真核宿主细胞直接投递到患者体内进行治疗；
    4)先通过体外转染技术将权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物与宿主细胞混合，然后将含有所述如权利要求1提供的gRNA序列或碱基编辑系统、权利要求8提供的非天然存在的或工程化的组合物的宿主细胞输回患者体内实施治疗；
    5)将权利要求10提供的真核宿主细胞输入患者体内实施治疗。
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