WO2019062522A1 - Arnsg, protéine cas9 modifiée, et kit - Google Patents

Arnsg, protéine cas9 modifiée, et kit 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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PCT/CN2018/104650
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English (en)
Chinese (zh)
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张芸
姜舒
纪惜銮
刘婕
郭明
罗朝霞
杨顺
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深圳三智医学科技有限公司
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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
    • 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
    • C12N15/87—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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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 RNAses, DNAses

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

La présente invention concerne un ARNsg, une protéine Cas9 modifiée, et un kit. Le kit comprend : un ARNsg ciblant spécifiquement un gène HBB ou un vecteur comprenant la séquence nucléotidique d'ARNsg; et/ou une enzyme Cas9 modifiée ou une séquence codant pour l'enzyme Cas9 modifiée ou un vecteur comprenant la séquence codant pour l'enzyme Cas9 modifiée; et/ou une séquence de gène donneur pour réparer un gène HBB de β-thalassémie ou un vecteur comprenant la séquence de gène donneur. L'application concerne en outre un applicateur du kit de clivage ou de réparation d'un gène HBB, ou plus spécifiquement pour la réparation d'un gène HBB de cellules souches hématopoïétiques autologues chez des patients présentant une β-thalassémie.
PCT/CN2018/104650 2017-09-30 2018-09-07 Arnsg, protéine cas9 modifiée, et kit WO2019062522A1 (fr)

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CN107630018B (zh) * 2017-09-30 2018-10-12 深圳三智医学科技有限公司 一种用于编辑或修复hbb基因的试剂盒
CN109486814A (zh) * 2017-10-31 2019-03-19 广东赤萌医疗科技有限公司 一种用于修复HBB1基因点突变的gRNA、基因编辑***、表达载体和基因编辑试剂盒
CN108795902A (zh) * 2018-07-05 2018-11-13 深圳三智医学科技有限公司 一种安全高效的CRISPR/Cas9基因编辑技术
GB201815670D0 (en) * 2018-09-26 2018-11-07 Univ Oxford Innovation Ltd Protein editing
CN109266651A (zh) * 2018-10-15 2019-01-25 广州鼓润医疗科技有限公司 基于CRISPR/Cas9技术编辑HBB-41/42缺失突变位点的sgRNA
CN111088253A (zh) * 2018-10-24 2020-05-01 广州鼓润医疗科技有限公司 针对hbb-28地中海贫血基因的crispr单碱基供体修复体系
CN112746071B (zh) * 2019-10-31 2022-01-04 华东师范大学 一种造血干细胞hbb基因修复的方法及产品
CN110951785A (zh) * 2019-12-30 2020-04-03 深圳三智医学科技有限公司 将CRISPR-Cas9***导入人干细胞的方法
CN112481304A (zh) * 2020-09-27 2021-03-12 镇江维根生物科技有限公司 一种多功能病毒载体的构建和应用
WO2022120786A1 (fr) * 2020-12-11 2022-06-16 The Third Affiliated Hospital Of Guangzhou Medical University Molécule de arng ciblant des exons du gène hbb2 de lapin, son procédé de synthèse, modèle de lapin ciblant le gène hbb2 et son procédé de construction
WO2022147759A1 (fr) * 2021-01-08 2022-07-14 Susheng Biotech (Hainan) Co., Ltd. Molécule d'arng ciblant l'intron i ou l'intron ii du gène hbb, son procédé de synthèse et procédé pour corriger les types de mutations du gène hbb
CN115141817B (zh) * 2021-03-30 2023-09-15 华东师范大学 一种细胞中hbb基因修复的方法及产品

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