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WO2019062522A1 - 一种sgRNA、改造的Cas9蛋白及试剂盒 - Google Patents

一种sgRNA、改造的Cas9蛋白及试剂盒 Download PDF

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WO2019062522A1
WO2019062522A1 PCT/CN2018/104650 CN2018104650W WO2019062522A1 WO 2019062522 A1 WO2019062522 A1 WO 2019062522A1 CN 2018104650 W CN2018104650 W CN 2018104650W WO 2019062522 A1 WO2019062522 A1 WO 2019062522A1
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hbb
gene
spcas9
sgrna
vector
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French (fr)
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张芸
姜舒
纪惜銮
刘婕
郭明
罗朝霞
杨顺
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Shenzhen Changene Medical Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
    • C12N15/90—Stable introduction of foreign DNA into chromosome
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]

Definitions

  • the present invention relates to a kit, and more particularly to an sgRNA, a modified Cas9 protein and a kit.
  • Thalassemia also referred to as thalassemia
  • marine anemia It is a common clinical hereditary hemolytic anemia. It is common in Southeast Asia, the Mediterranean and other regions. It is called thalassemia because it was first discovered in Italy, Greece, Malta and other regions along the Mediterranean coast.
  • Thalassemia is a hereditary hemoglobin disease caused by a gene deletion or mutation that regulates globin synthesis, resulting in an imbalance in the synthesis ratio of the alpha chain and beta chain globin that constitute hemoglobin, and a hemolytic anemia in which the life span of red blood cells is shortened.
  • Beta-thalassemia is a disorder of ⁇ -globin synthesis caused by mutations in the ⁇ -globin gene and is the most common monogenic genetic disease in the world.
  • CRISPR/Cas9 The emergence of the CRISPR/Cas9 system has made people aware of the power of gene editing tools in the body. Therefore, many scientists have recently studied how to use the CRISPR/Cas9 system to repair mutations in genetic diseases, so as to achieve the goal of treating single-gene genetic diseases. Research on beta-thalassemia is also included.
  • the object of the present invention is to provide a kit for editing or repairing a human hemoglobin gene (HBB gene) using a CRISPR/Cas9 system, which has strong targeting to the HBB gene and cleaves the HBB gene.
  • the efficiency is high and the off-target efficiency is low.
  • the invention provides a guide RNA (sgRNA) that functions to mediate specific binding of a Cas9 protein to a target gene; preferably, the target gene is a HBB gene.
  • sgRNA guide RNA
  • the nucleotide sequence encoding the sgRNA (that is, the DNA sequence corresponding to the sgRNA) is SEQ ID NO. 1, SEQ ID NO. 2, SEQ The sequence shown in ID NO. 3 or SEQ ID NO.
  • SEQ ID NO. 1 SEQ above
  • the sequence shown by ID NO.2, SEQ ID NO. 3 or SEQ ID NO. 4 is used to mediate the specific binding of the Cas9 protein to the HBB gene, has a relatively stronger targeting and is more tightly bound, and significantly enhances Cas9.
  • the invention provides a modified Cas9 protein, wherein the engineered Cas9 protein is mutated to lysine at position 848 of wild-type Cas9 protein to alanine and lysine at position 1003 Alanine and arginine at position 1060 are mutated to alanine;
  • the engineered Cas9 protein is mutated to alanine at position 848 of wild type Cas9 protein and arginine at position 925 to valine and lysine mutation at position 1003 Obtaining alanine and arginine at position 1060 to alanine;
  • the wild type Cas9 protein is a Streptococcus pyogenes Cas9 protein (SpCas9 protein), and the amino acid sequence thereof is shown in SEQ ID NO.
  • the surprising discovery of the present invention is that the modified Cas9 protein obtained by introducing the Cas9 protein into the above mutation site can more accurately recognize the HBB gene, thereby improving the efficiency of gene editing and reducing the off-target effect.
  • the invention also provides the use of the above sgRNA and/or engineered Cas9 protein for the preparation of a kit for editing or repairing the HBB gene.
  • the present invention provides a kit comprising: an oligonucleotide sequence encoding the above sgRNA or a vector comprising the oligonucleotide sequence.
  • the oligonucleotide sequence encoding the above sgRNA includes an sgRNA sense strand and an sgRNA antisense strand.
  • the sgRNA sense strand comprises as SEQ A nucleotide sequence represented by ID NO. 1, wherein the sgRNA antisense strand comprises the reverse complement of the nucleotide sequence set forth in SEQ ID NO.
  • the sgRNA sense strand comprises as SEQ The nucleotide sequence shown by ID NO. 2, wherein the sgRNA antisense strand comprises the reverse complement of the nucleotide sequence set forth in SEQ ID NO.
  • the sgRNA sense strand comprises as SEQ The nucleotide sequence shown by ID NO. 3, wherein the sgRNA antisense strand comprises the reverse complement of the nucleotide sequence set forth in SEQ ID NO.
  • the sgRNA sense strand comprises as SEQ The nucleotide sequence shown by ID NO. 4, wherein the sgRNA antisense strand comprises the reverse complement of the nucleotide sequence set forth in SEQ ID NO.
  • the vector comprising the oligonucleotide sequence encoding the above sgRNA may simultaneously contain a nucleotide sequence encoding the above-described engineered Cas9 protein.
  • the kit further comprises the engineered Cas9 protein, a nucleotide sequence encoding the engineered Cas9 protein, or a vector comprising a nucleotide sequence encoding the engineered Cas9 protein.
  • the vector comprising the nucleotide sequence encoding the engineered Cas9 protein may also contain an oligonucleotide sequence encoding the above sgRNA.
  • the kit further comprises a donor gene sequence for repairing the HBB gene or a vector comprising the donor gene sequence.
  • the donor gene sequence for repairing the HBB gene includes a wild-type HBB gene sequence
  • the donor gene sequence for repairing the HBB gene may contain a non-homologous sequence of a wild-type HBB gene flanked by homologous sequences of two wild-type HBB genes for the HBB gene.
  • HDR homologous-mediated double-stranded DNA repair
  • the invention also provides the use of the above kit for cleavage of the HBB gene or repair of the HBB gene.
  • the application is to repair the HBB gene of autologous hematopoietic stem cells of ⁇ -thalassemia patients.
  • Repairing the HBB gene of autologous hematopoietic stem cells from patients with beta-thalassemia can transform autologous hematopoietic stem cells from patients with beta-thalassemia into hematopoietic stem cells with normal beta-globin function.
  • the method of application is: an oligonucleotide sequence of the above sgRNA or a vector comprising the oligonucleotide sequence, and/or a nucleotide sequence encoding the engineered Cas9 protein or comprising the modification encoding the modification
  • a vector for the nucleotide sequence of the Cas9 protein, and/or a vector comprising the above-described donor gene sequence for repairing the HBB gene is introduced into the recipient cell to complete editing or repair of the HBB gene.
  • the method for introducing the recipient cell can utilize a introduction method commonly used in the art, such as microinjection, liposome-mediated, viral transfection, electrotransfection, etc.; wherein the virus transfection method
  • adeno-associated virus vector (Adeno-associated) is utilized Virus, AAV) system.
  • the kit provided by the invention has strong targeting to the HBB gene, high efficiency of cutting the HBB gene, low off-target effect, and can effectively repair the HBB gene, especially the HBB gene of autologous hematopoietic stem cells of ⁇ -thalassemia patients. It has broad prospects in clinical research and therapeutic applications.
  • the target site of the sgRNA in the kit provided by the present invention is designed in the intron region of the HBB gene, and after the intron is cleavage, the donor carrying the normal HBB gene is recombined into the cell by homologous recombination technology. In the genome, repair of downstream gene mutations. Since gene editing is not performed by cutting exons, the risk of off-target effects of gene editing technology is reduced compared with previous gene editing.
  • the engineered Cas9 enzyme in the kit provided by the invention can improve gene editing targeting and target gene cleavage efficiency, reduce off-target effect, and reduce gene mutation caused by off-target.
  • FIG. 1 Schematic diagram of the structure of the psgRNA-HBB-SpCas9-SZ vector.
  • U6 is the promoter driving sgRNA
  • CAG is CMV early enhancer/chicken ⁇
  • the actin (CAG) promoter is used to drive expression of Cas9-SZ.
  • FIG. 1 Electrophoresis results of T7 endonuclease I digestion assay for psgRNA-HBB-SpCas9-SZ insertion/deletion efficiency.
  • Lane 1 is a control group without sgRNA
  • lanes 2 to 6 are psgRNA1-HBB-SpCas9-SZ1, psgRNA2-HBB-SpCas9-SZ1, psgRNA3-HBB-SpCas9-SZ1, psgRNA4-HBB-SpCas9-SZ1, and psgRNA4- The result of digestion of HBB-SpCas9-SZ2.
  • Figure 3 Statistical results of psgRNA-HBB-SpCas9-SZ insertion/deletion efficiency by T7 endonuclease I digestion. 1-5 are statistical results of psgRNA1-HBB-SpCas9-SZ1, psgRNA2-HBB-SpCas9-SZ1, psgRNA3-HBB-SpCas9-SZ1, psgRNA4-HBB-SpCas9-SZ1, and psgRNA4-HBB-SpCas9-SZ2, respectively.
  • Figure 4A The sequence of HBB-sgRNA4-S was aligned with the human genome sequence, and the three closest target sequences were selected.
  • Figure 4B shows that the psgRNA4-HBB-SpCas9-SZ1 has no cleavage at these sites.
  • Figure 5A is a structural diagram of the pAAV-sgRNA4-HBB-EF1mini-EGFP vector.
  • FIG. 5B AAV virus packaging process.
  • Figure 5C Fluorescent picture of HEK293 cells infected with packaged AAV virus.
  • Figure 6A is a structural diagram of the pAAV-sgRNA4-HBB-EF1mini-SpCas9-SZ1 vector and the pAAV-HBB-Donor vector.
  • Figure 6B Fluorescence picture of EGFP expressed by recombinant cells.
  • Figure 6C Results of genotypic sequencing after homologous recombination repair, wherein the red marker region is the HBB gene homology arm sequence, the blue region is the EF1mini promoter sequence, and the cyan region is the EGFP gene sequence.
  • test methods used in the following examples are all conventional methods unless otherwise specified.
  • SpCas9-SZ1 protein Mutation of the 848th lysine of the SpCas9 protein (GI: 81533697) to alanine and the mutation of the 1003th lysine to alanine and the 1060th arginine to alanine
  • the engineered SpCas9 protein was named SpCas9-SZ1 protein.
  • SpCas9-SZ2 protein Mutation of the 848th lysine of the SpCas9 protein (GI: 81533697) to alanine and the 925th arginine to valine and the 1003th lysine to alanine and the 1060th The arginine was mutated to alanine, and the engineered SpCas9 protein obtained was named as SpCas9-SZ2 protein.
  • HBB-sgRNA1-4 Four sets of sgRNA sequences (HBB-sgRNA1-4) were synthesized, and the four sets of sgRNA sequences respectively included the nucleotide sequence shown in SEQ ID NO. 1-4 or its reverse complement.
  • the naming and nucleotide sequences of the four sgRNA sequences (HBB-sgRNA1-4) are shown in Table 1: wherein each sgRNA sequence consists of two sequences, one of which is the sense strand DNA (S) and the other is antisense. Strand DNA (AS).
  • sgRNA-HBB-SpCas9-SZ1 vector The sequence of sgRNA was cloned into pSpCas9-SZ1 to obtain a recombinant vector in which sgRNA and SpCas9-SZ1 protein were co-expressed: psgRNA-HBB-SpCas9-SZ1 vector.
  • the sequence of the sgRNA was cloned into pSpCas9-SZ2 to obtain a recombinant vector in which sgRNA and SpCas9-SZ2 protein were co-expressed: psgRNA-HBB-SpCas9-SZ2 vector.
  • FIG. 1 A schematic diagram of the structure of the psgRNA-HBB-SpCas9-SZ1 and psgRNA-HBB-SpCas9-SZ2 recombinant vectors is shown in FIG. Among them, U6 is the promoter that drives sgRNA, CAG is CMV early The enhancer/chicken beta actin (CAG) promoter is used to drive expression of the SpCas9-SZ1 or SpCas9-SZ2 protein.
  • CAG enhancer/chicken beta actin
  • the annealing conditions were: 37 ° C, 30 min; 95 ° C, 5 min; at a rate of 5 ° C ⁇ min -1 to 25 ° C.
  • the annealed product was named HBB-sgRNA1.
  • the enzyme was digested at 37 ° C for 1 h.
  • T4 ligase (T4 ligase) 1
  • HBB-SpCas9-SZ2 vector was identical to the construction of psgRNA1-HBB-SpCas9-SZ1 described above.
  • Sequencing results showed that psgRNA2-HBB-SpCas9-SZ1, psgRNA3-HBB-SpCas9-SZ1, psgRNA4-HBB-SpCas9-SZ1, psgRNA1-HBB-SpCas9-SZ2, psgRNA2-HBB-SpCas9-SZ2, psgRNA3-HBB-SpCas9- The SZ2, psgRNA4-HBB-SpCas9-SZ2 vectors were all successfully constructed.
  • the psgRNA-HBB-SpCas9-SZ vector referred to below is psgRNA1-HBB-SpCas9-SZ1, psgRNA2-HBB-SpCas9-SZ1, psgRNA3-HBB-SpCas9-SZ1, psgRNA4-HBB-SpCas9-SZ1, psgRNA1-HBB-SpCas9- A general term for SZ2, psgRNA2-HBB-SpCas9-SZ2, psgRNA3-HBB-SpCas9-SZ2, psgRNA4-HBB-SpCas9-SZ2 vector.
  • HEK293T cells were seeded in a 24-well plate at 1.5 ⁇ 10 5 cells/well with a total cell suspension volume of 500 ⁇ l;
  • the insertion/deletion efficiency was determined by T7 endonuclease I digestion to verify the cleavage efficiency of hp gene by psgRNA-HBB-SpCas9-SZ:
  • transfected cells were harvested; centrifuged at 400 g for 5 min; the supernatant was discarded and the cells were resuspended in 1 x PBS.
  • the cell genomic DNA was extracted using a cellular genomic DNA extraction kit.
  • the specific forward primer of the HBB gene is the nucleotide sequence shown in SEQ ID NO. 13; specificity The reverse primer is the nucleotide sequence shown in SEQ ID NO.
  • the PCR amplification procedure was: 95 ° C, 3 min; 95 ° C, 30 s, 66 ° C, 30 s, 72 ° C, 30 s, 26 cycles; 72 ° C, 5 min.
  • the DNA fragment of interest is recovered using a gel recovery kit.
  • T7 endonuclease I 1 ⁇ l was added to the above annealed product, and the mixture was incubated at 37 ° C for 30 min.
  • the digested product was electrophoresed on a 2% agarose gel; the ratio of the cleavage of the HBB gene by the psgRNA-HBB-SpCas9-SZ vector was detected by comparing the ratio of the fragment digested with T7 endonuclease I and not digested.
  • lane 1 is a control group without sgRNA
  • lanes 2 to 6 are psgRNA1-HBB-SpCas9-SZ1, psgRNA2-HBB-SpCas9-SZ1, psgRNA3-HBB-SpCas9-SZ1, psgRNA4-
  • HBB-sgRNA4-S was aligned with the human genome sequence, and the closest target was selected (Fig. 4A). We designed specific primers for PCR amplification of these target regions, and then The amplified products were sequenced and the sequencing results showed that the psgRNA4-HBB-SpCas9-SZ1 vector was not cleaved at these sites (Fig. 4B). That is, the psgRNA4-HBB-SpCas9-SZ1 vector constructed by using the sgRNA sequence provided by the kit of the present invention and the modified Cas9 protein has no off-target, has high specificity and high safety.
  • Adeno-associated virus is one of the most commonly used viral vectors in gene therapy, but its limited loading capacity limits its packaging of the CRISPR-Cas9 system for future applications.
  • the mini promoter is only 500 bp in size and its nucleotide sequence is Seq As shown in ID NO. 16, it can well drive the expression of Cas9.
  • the EF1mini promoter was synthesized by chemical synthesis and ligated into the psgRNA-HBB-SpCas9-SZ vector. To verify the activity of the EF1 mini promoter, the promoter was used to drive the expression of the reporter gene EGFP to construct the pAAV-sgRNA4-HBB-EF1mini-EGFP vector.
  • the vector structure is shown in Figure 5A.
  • the AAV virus was packaged by a three-plasmid co-transfection method using HEK293T cells, as shown in Fig. 5B.
  • HEK293T cells were seeded in a 24-well plate at 1.5 x 105 cells/well with a total cell suspension volume of 500 ⁇ l. After 24 hours, the AAV virus suspension was added for cell infection.
  • Figure 5C is a fluorescent photograph of HEK293T cells infected with different concentrations of AAV virus 96 h after infection.
  • the vector obtained by PCR amplification of the SpCas9-SZ1 gene in place of the EGFP gene of the pAAV-sgRNA4-HBB-EF1mini-EGFP vector described in the above step 1-(1) is the pAAV-sgRNA1-HBB-EF1mini-SpCas9-SZ1 vector.
  • a schematic structural view of the carrier is shown in Fig. 6A.
  • the HBB homology arm was amplified by PCR, inserted into both sides of the EF1mini-EGFP sequence of the pAAV-EF1mini-EGFP vector described in the above step 1-(1), and the entire sequence was placed in the AAV vector to obtain pAAV-HBB-
  • the Donor vector, pAAV-HBB-Donor vector structure is shown in Figure 6A.
  • the HEK293T cells were infected with the viruses of pAAV-HBB-Donor and pAAV-sgRNA4-HBB-EF1mini-SpCas9-SZ1 packaged by the AAV virus packaging method described in the above steps 1-(2).
  • a fluorescent picture of EGFP expressed by the recombined cells is shown in Figure 6B.
  • EGFP-positive cells were sorted by flow cytometry, and the genomic DNA of the cells was extracted, and then the sequence between the homologous arms was amplified by specific primers, and the PCR products were sent for sequencing.
  • the sequencing results are shown in Figure 6C.
  • the red marker region is the HBB gene homology arm sequence
  • the blue region is the EF1 promoter sequence
  • the cyan region is the EGFP gene sequence. The results showed that the HBB gene homology arm sequence was successfully recombined into the recipient cells.

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Abstract

提供了一种sgRNA、改造的Cas9蛋白及试剂盒。该试剂盒包括：特异性靶向HBB基因的sgRNA或包含该sgRNA核苷酸序列的载体；和/或改造的Cas9酶或编码该改造的Cas9酶的序列或包含编码该改造的Cas9酶序列的载体；和/或用于修复β-地中海贫血HBB基因的供体基因序列或包含所述供体基因序列的载体。还提供了所述试剂盒在切割或修复HBB基因中的应用，尤其是在修复β-地中海贫血患者自体造血干细胞的HBB基因中的应用。

Description

一种sgRNA、改造的Cas9蛋白及试剂盒 技术领域
本发明涉及一种试剂盒，尤其是涉及一种sgRNA、改造的Cas9蛋白及试剂盒。
 背景技术
地中海贫血（Thalassemia，简称地贫）又称海洋性贫血。是临床常见的遗传性溶血性贫血。在东南亚、地中海等地区多见，因最早在地中海沿岸的意大利、希腊、马耳他等地区发现，故称地中海贫血。
地中海贫血是一种遗传性血红蛋白病，是由于调控珠蛋白合成的基因缺失或突变，导致构成血红蛋白的α链和β链珠蛋白的合成比例失衡，红细胞寿命缩短的一种溶血性贫血。
根据基因缺陷的分类，临床上主要分为α链珠蛋白地中海贫血及β链珠蛋白地中海贫血。β-地中海贫血是由β-珠蛋白基因突变导致的β-珠蛋白合成障碍，是全球最常见的单基因遗传病。
2008年WHO全球血红蛋白病流行病学报告：在229个国家中约占71%人群存在血红蛋白病这一重大健康问题。在这些国家出生的新生儿占全球新生儿的89%，其中每年超过330000例的新生儿与血红蛋白病有关（其中83%为镰状细胞症，17%为地中海贫血）。5岁以下儿童死亡者中血红蛋白病约占3.4%。每年约有56000例重型β-地中海贫血，其中至少有30000例需规范输血才能存活，5500例重型α-地中海贫血死于围生期。目前国内外对地中海贫血治疗主要方法：规范性长期输血和去铁治疗；根治方法：HLA相合的造血干细胞移植；姑息方法：脾切除术等。但这些治疗方法给病人家庭及社会带来了沉重的负担。
随着分子生物学和分子遗传学理论和技术的迅速发展，人们对基因治疗进行了广泛和深入的基础研究，并在实施方法、转移效率、基因表达、动物试验、安全性评价等方面取得了很大的进展，基因治疗作为一种临床治疗方法已逐渐开始为人们所接受。基因治疗β-地中海贫血作为新的治疗方式将患者有缺陷的基因进行替代或补偿缺陷功能成为新的研究热点。
CRISPR/Cas9系统的出现使人们认识到体内基因编辑工具的强大，因此最近许多科学家研究如何利用CRISPR/Cas9系统修复遗传疾病致病基因的突变，从而达到治疗单基因遗传病的目的。对β-地中海贫血的研究也在此之列。
然而，作为基因编辑技术，脱靶效应是不可避免的一个缺点，影响了其临床转化应用的进程。因此，如何在提高CRISPR/Cas技术基因编辑效率的同时提高其靶向性，是利用CRISPR/Cas技术修复β-地中海贫血患者的HBB基因面临的一大难题。
 技术问题
为弥补现有技术的不足，本发明的目的在于提供一种试剂盒，该试剂盒利用CRISPR/Cas9系统编辑或修复人类血红蛋白基因（HBB基因），对HBB基因的靶向性强，切割HBB基因的效率高，脱靶效率低。
 技术解决方案
一方面，本发明提供了一种向导RNA（sgRNA），其作用是介导Cas9蛋白与靶基因特异性结合；所述的靶基因优选为HBB基因。
编码所述的sgRNA的核苷酸序列（也就是所述的sgRNA对应的DNA序列）为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的序列。
本发明意外的发现，上述SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的序列用于介导Cas9蛋白与HBB基因特异性结合，具有相对更强的靶向性并且结合更加紧密，显著提高了Cas9蛋白对HBB基因的切割效率，并明显减少了脱靶效应。
另一个方面，本发明提供了一种改造的Cas9蛋白，所述的改造的Cas9蛋白为将野生型Cas9蛋白第848位赖氨酸突变成丙氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸获得；
优选地，所述的改造的Cas9蛋白为将野生型Cas9蛋白第848位赖氨酸突变成丙氨酸及第925位精氨酸突变成脯氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸获得；
优选地，所述的野生型Cas9蛋白为为化脓性链球菌Cas9蛋白（SpCas9蛋白），其氨基酸序列如SEQ ID NO.15所示。
本发明意外的发现，上述突变位点引入Cas9蛋白后得到的改造后的Cas9蛋白能够更加精准地识别HBB基因，从而提高基因编辑效率，降低脱靶效应。
本发明还提供了上述sgRNA和/或改造的Cas9蛋白在制备编辑或修复HBB基因的试剂盒中的应用。
再一个方面，本发明提供了一种试剂盒，该试剂盒包括：编码上述sgRNA的寡核苷酸序列或包含该寡核苷酸序列的载体。
进一步地，编码上述sgRNA的寡核苷酸序列包括sgRNA正义链和sgRNA反义链。
在一个优选的实施方案中，所述的sgRNA正义链包括如SEQ ID NO.1所示的核苷酸序列，所述的sgRNA反义链包括如SEQ ID NO.1所示的核苷酸序列的反向互补序列。
在另一个优选的实施方案中，所述的sgRNA正义链包括如SEQ ID NO.2所示的核苷酸序列，所述的sgRNA反义链包括如SEQ ID NO.2所示的核苷酸序列的反向互补序列。
在又一个优选的实施方案中，所述的sgRNA正义链包括如SEQ ID NO.3所示的核苷酸序列，所述的sgRNA反义链包括如SEQ ID NO.3所示的核苷酸序列的反向互补序列。
在再一个优选的实施方案中，所述的sgRNA正义链包括如SEQ ID NO.4所示的核苷酸序列，所述的sgRNA反义链包括如SEQ ID NO.4所示的核苷酸序列的反向互补序列。
更进一步地，所述的包含编码上述sgRNA的寡核苷酸序列的载体上可以同时含有编码上述改造的Cas9蛋白的核苷酸序列。
优选地，所述的试剂盒还包括上述改造的Cas9蛋白、编码该改造的Cas9蛋白的核苷酸序列或包含编码该改造的Cas9蛋白的核苷酸序列的载体。
在一个优选的实施方案中，所述的包含编码改造的Cas9蛋白的核苷酸序列的载体上可以同时含有编码上述sgRNA的寡核苷酸序列。
更优选地，所述的试剂盒还包括用于修复HBB基因的供体基因序列或包含该供体基因序列的载体。
进一步地，所述的用于修复HBB基因的供体基因序列包括野生型HBB基因序列；
优选地，所述的用于修复HBB基因的供体基因序列可含有野生型HBB基因的非同源序列，该非同源序列侧接两个野生型HBB基因的同源序列，以便在HBB基因处有效地进行HDR（同源介导的双链DNA修复）。
本发明还提供了上述试剂盒在切割HBB基因或修复HBB基因中的应用。
进一步地，所述应用为修复β-地中海贫血患者自体造血干细胞的HBB基因。修复β-地中海贫血患者自体造血干细胞的HBB基因可将β-地中海贫血患者自体造血干细胞转变为具有正常合成β-珠蛋白功能的造血干细胞。
进一步地，所述应用的方法为：将上述sgRNA的寡核苷酸序列或包含该寡核苷酸序列的载体，和/或编码该改造的Cas9蛋白的核苷酸序列或包含编码该改造的Cas9蛋白的核苷酸序列的载体，和/或包含上述用于修复HBB基因的供体基因序列的载体导入受体细胞，以完成编辑或修复HBB基因。
其中，所述导入受体细胞的方法可以利用本领域常用的导入方法，例如显微注射法、脂质体介导法、病毒转染法、电转染等；其中，所述病毒转染法优选利用腺相关病毒载体（Adeno-associated Virus, AAV）系统。
 有益效果
本发明的有益效果在于：
1、本发明提供的试剂盒，靶向HBB基因的靶向性强，切割HBB基因的效率高，脱靶效应低，且可有效修复HBB基因，尤其是β-地中海贫血患者自体造血干细胞的HBB基因，在临床研究及治疗应用上前景广阔。
2、本发明提供的试剂盒中的sgRNA的靶位点设计在HBB基因的内含子区域，剪切该内含子后利用同源重组技术，将携带有正常HBB基因供体重组到细胞的基因组中，修复下游基因突变。因不用通过剪切外显子来进行基因编辑，故同以往的基因编辑相比，降低了基因编辑技术脱靶效应导致的风险。
3、本发明提供的试剂盒中的改造的Cas9酶可提高基因编辑靶向性和靶基因切割效率，减少脱靶效应，减少脱靶导致的基因突变。
 附图说明
图1 psgRNA-HBB-SpCas9-SZ载体结构示意图。U6为驱动sgRNA的启动子，CAG为CMV early enhancer/chicken β actin (CAG)启动子，用于驱动Cas9-SZ的表达。
图2 T7 endonucleaseⅠ酶切法检测psgRNA-HBB-SpCas9-SZ插入/缺失效率的电泳结果。泳道1为未含sgRNA的对照组，泳道2~6分别为psgRNA1-HBB-SpCas9-SZ1、psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1和psgRNA4-HBB-SpCas9-SZ2的酶切结果。
图3 T7 endonucleaseⅠ酶切法检测psgRNA-HBB-SpCas9-SZ插入/缺失效率的统计结果。1-5分别为psgRNA1-HBB-SpCas9-SZ1、psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1和psgRNA4-HBB-SpCas9-SZ2的统计结果。Data = Mean±SE , n =3。
图4A HBB-sgRNA4-S的序列和人类基因组序列进行比对，从中挑选的3个最为接近的靶点序列。
图4B 测序结果表明，psgRNA4-HBB-SpCas9-SZ1在这些位点上都无剪切。
图5A pAAV-sgRNA4-HBB-EF1mini-EGFP载体结构图。
图5B AAV病毒包装过程。
图5C 包装好的AAV病毒感染HEK293细胞的荧光图片。
图6A pAAV-sgRNA4-HBB-EF1mini-SpCas9-SZ1载体和pAAV-HBB-Donor载体结构图。
图6B 重组后的细胞表达EGFP的荧光图片。
图6C 同源重组修复后的基因型测序结果，其中，红色标记区域为HBB基因同源臂序列，蓝色区域为EF1mini启动子序列，青绿色区域为EGFP基因序列。
 本发明的实施方式
下述实施例中所使用的试验方法如无特殊说明，均为常规方法。
下述实施例中所使用的材料、试剂等，如无特殊说明，均可从商业途径得到。以下实施例便于更好地理解本发明，但并不限定本发明。
实验例1
验证该试剂盒对HBB基因的切割效率
1、改造SpCas9蛋白
将SpCas9蛋白（GI:81533697）的第848位赖氨酸突变成丙氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸，获得的改造后的SpCas9蛋白命名为SpCas9-SZ1蛋白。
将SpCas9蛋白（GI:81533697）的第848位赖氨酸突变成丙氨酸及第925位精氨酸突变成脯氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸，获得的改造后的SpCas9蛋白命名为SpCas9-SZ2蛋白。
2、构建pSpCas9-SZ（BB）载体
突变pSpCas9(BB)（Addgene plasmid ID: 42230）载体中SpCas9的编码序列，使其编码步骤1所述的SpCas9-SZ1酶，新载体命名为pSpCas9-SZ1载体。
突变pSpCas9(BB)（Addgene plasmid ID: 42230）载体中SpCas9的编码序列，使其编码步骤1所述的SpCas9-SZ2酶，新载体命名为pSpCas9-SZ2载体。
3、合成特异性靶向HBB基因的sgRNA寡核苷酸序列
合成4组sgRNA序列（HBB-sgRNA1-4），这四组sgRNA序列分别包括如SEQ ID NO.1-4所示的核苷酸序列或其反向互补序列。这四组sgRNA序列（HBB-sgRNA1-4）的命名及核苷酸序列分别见表1：其中，每组sgRNA序列包括两条序列，其中一条为正义链DNA（S），另一条为反义链DNA（AS）。
表1
Figure 495831dest_path_image001
4、构建psgRNA-HBB-SpCas9-SZ1和psgRNA-HBB-SpCas9-SZ2载体
将sgRNA的序列克隆到pSpCas9-SZ1中，获得sgRNA和SpCas9-SZ1蛋白共表达的重组载体：psgRNA-HBB-SpCas9-SZ1载体。
将sgRNA的序列克隆到pSpCas9-SZ2中，获得sgRNA和SpCas9-SZ2蛋白共表达的重组载体：psgRNA-HBB-SpCas9-SZ2载体。
psgRNA-HBB-SpCas9-SZ1和psgRNA-HBB-SpCas9-SZ2重组载体的结构示意图见图1。其中，U6为驱动sgRNA的启动子，CAG为CMV early enhancer/chicken β actin (CAG)启动子，用于驱动SpCas9-SZ1或SpCas9-SZ2蛋白的表达。
以psgRNA1-HBB-SpCas9-SZ1载体的构建方法为例描述psgRNA-HBB-SpCas9-SZ1和psgRNA-HBB-SpCas9-SZ2重组载体的构建：
（1）配置上述步骤3所述的第1组sgRNA的正义链HBB-sgRNA1-S（SEQ ID NO.5）和第1组sgRNA的反义链HBB-sgRNA1-As（SEQ ID NO.6）的悬液，使其终浓度分别为100 µM，按下述体系进行退火：
组分体积（µl）
HBB-sgRNA1-S                                                                 1
HBB-sgRNA1-AS                                                               1
10×T4 ligation buffer                                                      1
T4多聚核苷酸激酶（T4 PNK）                                1
ddH 2O                                                                                          6；
其中，退火条件为：37℃，30 min；95 ℃，5 min；以5℃·min-1的速度降至 25℃。退火后的产物命名为HBB-sgRNA1。
（2）将上述步骤4-（1）获得的HBB-sgRNA1用ddH 2O按1:200稀释至总体积为200 µl。
（3）用BbSI酶酶切步骤2获得的pSpCas9-SZ1载体，所用酶切体系如下：
组分体积（µl）
pSpCas9-SZ1（1 µg/µl）                                     1
10×NEB buffer                                                                                     2
FastDigest BbsI                                                                                            1
ddH 2O                                                                                                             16；
37℃酶切反应1 h。
（4）将步骤4-（2）获得的稀释后的HBB-sgRNA1连接入BbSI酶酶切后的pSpCas9-SZ1载体中：
在步骤（3）所用酶切体系的基础上添加下述组分，
组分体积（µl）
步骤4-（2）的产物                                   1
10×T4 ligase buffer                                       2.5
T4连接酶（T4 ligase）                             1
ddH 2O                                                                        0.5；
25℃连接30 min。
（5）鉴定克隆：将上述步骤4-（4）的连接产物取2µl热转化至Stbl3感受态细胞中，涂布平板，37℃过夜培养。挑取单克隆，37℃摇菌过夜，抽提质粒并进行测序，鉴定HBB-sgRNA1是否插入pSpCas9-SZ1载体的U6启动子和CAG启动子之间。测序结果显示，HBB-sgRNA1正确插入。获得的新载体命名为psgRNA1-HBB-SpCas9-SZ1。
psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1、psgRNA1-HBB-SpCas9-SZ2、psgRNA2-HBB-SpCas9-SZ2、psgRNA3-HBB-SpCas9-SZ2、psgRNA4-HBB-SpCas9-SZ2载体的构建方法同上述psgRNA1-HBB-SpCas9-SZ1的构建。
测序结果显示，psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1、psgRNA1-HBB-SpCas9-SZ2、psgRNA2-HBB-SpCas9-SZ2、psgRNA3-HBB-SpCas9-SZ2、psgRNA4-HBB-SpCas9-SZ2载体均构建成功。
以下所称psgRNA-HBB-SpCas9-SZ载体为psgRNA1-HBB-SpCas9-SZ1、psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1、psgRNA1-HBB-SpCas9-SZ2、psgRNA2-HBB-SpCas9-SZ2、psgRNA3-HBB-SpCas9-SZ2、psgRNA4-HBB-SpCas9-SZ2载体的统称。
5、共表达重组载体转染HEK293T细胞：
按1.5×105 个细胞/孔将HEK293T细胞接种于24孔板中，细胞悬液总体积为500 µl；
种板24h后进行细胞转染：（1）用无血清培养基将2 µg psgRNA-HBB-SpCas9-SZ载体和1µg EGFP载体稀释至250µl，温和混匀；（2）用培养基将脂质体稀释至250µl，室温孵育5min；（3）将上述5-（1）步骤和5-（2）步骤获取的混合物混匀，室温孵育20min；（4）吸去HEK293T细胞中的培养液，将步骤5-（3）获得的混合物加入细胞，细胞置于细胞恒温培养箱37℃孵育6 h后换成含10 % FBS的DMEM培养基，转染48h后观察转染效率。
6、T7 endonucleaseⅠ酶切法检测插入/缺失效率，以验证psgRNA-HBB-SpCas9-SZ对HBB基因切割效率：
（1）收取转染后的细胞；400g离心5min；弃去上清液，用1×PBS重悬细胞。
利用细胞基因组DNA提取试剂盒提取其细胞基因组DNA。
（2）以基因组DNA为模板，利用针对HBB基因的特异性引物按如下程序进行PCR扩增；所述HBB基因的特异性正向引物为SEQ ID NO.13所示核苷酸序列；特异性反向引物为SEQ ID NO.14所示核苷酸序列；
所述PCR扩增程序为：95℃，3 min；95℃，30 s，66℃，30 s，72℃，30 s，26个循环；72℃，5 min。
（3）扩增产物于1.5%琼脂糖凝胶中进行电泳；
利用胶回收试剂盒回收目的DNA片段。
（4）取步骤（3）所得纯化的DNA 17 µl，加入2 µlT7核酸内切酶Ⅰ（T7 endonucleaseⅠ）缓冲液，按下述程序进行变性退火处理：
步骤温度变温速率时间
变性                            95 ℃                                                                        5 min
退火                            95-85 ℃          -2 ℃/s   
                                     85-25 ℃          -0.1 ℃/s
维持温度                   4 ℃                                                                           Hold；
于上述退火产物中加入1 µl T7 endonucleaseⅠ，混匀后37℃孵育酶切30 min。
（5）酶切产物于2%琼脂糖凝胶中电泳；比较被T7 endonucleaseⅠ酶切及未被酶切的片段的比例即可检测出psgRNA-HBB-SpCas9-SZ载体对HBB基因的切割效率。电泳结果见图2，其中，泳道1为未含sgRNA的对照组，泳道2~6分别为psgRNA1-HBB-SpCas9-SZ1、psgRNA2-HBB-SpCas9-SZ1、psgRNA3-HBB-SpCas9-SZ1、psgRNA4-HBB-SpCas9-SZ1和psgRNA4-HBB-SpCas9-SZ2的酶切结果。
psgRNA-HBB-SpCas9-SZ载体酶切效率的统计结果见图3。结果表明，本发明的试剂盒中所提供的4组sgRNA均有较好的编辑效率，其中以第4组sgRNA的编辑效率最高；在sgRNA序列相同的情况下，SpCas9-SZ2蛋白的酶切效率略优于，SpCas9-SZ1蛋白。
实验例2
验证该试剂盒的脱靶效率
1、将HBB-sgRNA4-S的序列和人类基因组序列进行比对，从中挑选了与其最为接近的靶点（图4A），我们针对这些靶点区域设计特异性的引物进行PCR扩增，然后对扩增产物进行测序，测序结果表明，psgRNA4-HBB-SpCas9-SZ1载体在这些位点上都无剪切（图4B）。即利用本发明试剂盒提供的sgRNA序列和改造后的Cas9蛋白构建的psgRNA4-HBB-SpCas9-SZ1载体无脱靶，特异性强，安全性高。
实验例3
验证该试剂盒的基因重组效率
1、验证EF1迷你（EF1mini）启动子的驱动效率
腺相关病毒是目前基因治疗中最常用的病毒载体之一，但其装载容量有限，限制了其包装CRISPR-Cas9系统用于将来的应用之中。为此我们对启动Cas9的启动子进行了改造，采用了新型的EF1mini启动子用于我们的系统当中。该迷你启动子大小仅为500 bp，其核苷酸序列如Seq ID NO.16所示，能够很好的驱动Cas9的表达。
（1）用化学合成方法，合成EF1mini启动子，将其连接入psgRNA-HBB-SpCas9-SZ载体。为验证该EF1迷你启动子的活性，先用该启动子驱动报告基因EGFP的表达，构建pAAV-sgRNA4-HBB-EF1mini-EGFP载体，载体结构图见图5A。
（2）利用HEK293T细胞，通过三质粒共转染方法，包装出AAV病毒，如图5B所示。
（3）包装好的AAV病毒感染HEK293T细胞：
按1.5×105 个细胞/孔将HEK293T细胞接种于24孔板中，细胞悬液总体积为500µl。种板24h后加入AAV病毒悬液进行细胞感染。图5C所示为感染后96h，不同浓度的AAV病毒感染HEK293T细胞的荧光照片。
该结果表明，该EF1迷你启动子能够很好地驱动目的基因的表达。
2、构建pAAV-sgRNA4-HBB-EF1mini-SpCas9-SZ1腺相关病毒载体
用PCR扩增SpCas9-SZ1基因，替代上述步骤1-（1）所述pAAV-sgRNA4-HBB-EF1mini-EGFP载体的EGFP基因后的载体即为pAAV-sgRNA1-HBB-EF1mini-SpCas9-SZ1载体，该载体的结构示意图见图6A。
3、构建包含用于修复β-地中海贫血HBB基因的供体基因序列的载体：pAAV-HBB-Donor载体
用PCR扩增HBB同源臂，插入至上述步骤1-（1）所述pAAV-EF1mini-EGFP载体的EF1mini-EGFP序列的两侧后，将整个序列放入AAV载体中，得到pAAV-HBB-Donor载体，pAAV-HBB-Donor载体结构图见图6A。
4、以上述步骤1-（2）所述AAV病毒包装方法包装的pAAV-HBB-Donor和pAAV-sgRNA4-HBB-EF1mini-SpCas9-SZ1的病毒感染HEK293T细胞。重组后的细胞表达EGFP的荧光图片见图6B。
5、分析同源重组修复后的基因型：
以流式细胞仪分选EGFP阳性的细胞，提取细胞基因组DNA，然后以特异性引物扩增同源臂之间的序列，将PCR产物送测序。测序结果见图6C。其中，红色标记区域为HBB基因同源臂序列，蓝色区域为EF1启动子序列，青绿色区域为EGFP基因序列。结果显示，HBB基因同源臂序列被成功重组到了受体细胞中。

Claims (10)

  1. 一种sgRNA，编码该sgRNA的核苷酸序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3或SEQ ID NO.4所示的序列。
  2. 一种改造的Cas9蛋白，该改造的Cas9蛋白为将野生型Cas9蛋白第848位赖氨酸突变成丙氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸获得。
  3. 一种改造的Cas9蛋白，该改造的Cas9蛋白为将野生型Cas9蛋白第848位赖氨酸突变成丙氨酸及第925位精氨酸突变成脯氨酸及第1003位赖氨酸突变成丙氨酸及第1060位精氨酸突变成丙氨酸获得。
  4. 如权利要求2或3所述的改造的Cas9蛋白，其中，所述的野生型Cas9蛋白为氨基酸序列如SEQ ID NO.15所示的SpCas9蛋白。
  5. 权利要求1所述的sgRNA和/或权利要求2或3所述的改造的Cas9蛋白在制备编辑或修复HBB基因的试剂盒中的应用。
  6. 一种试剂盒，该试剂盒包括：编码权利要求1所述的sgRNA的寡核苷酸序列或包含该寡核苷酸序列的载体。
  7. 如权利要求6所述的试剂盒，该试剂盒还包括：权利要求2或3所述的改造的Cas9蛋白或编码该改造的Cas9蛋白的核苷酸序列或包含编码该改造的Cas9蛋白的核苷酸序列的载体。
  8. 如权利要求6所述的试剂盒，该试剂盒还包括：用于修复HBB基因的供体基因序列或包含该供体基因序列的载体。
  9. 权利要求6-8任一项所述的试剂盒在切割HBB基因或修复HBB基因中的应用。
  10. 权利要求6-8任一项所述的试剂盒在修复β-地中海贫血患者自体造血干细胞的HBB基因中的应用。
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