WO2016197359A1 - Procédé d'inactivation spécifique du gène sla-1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-1 - Google Patents

Procédé d'inactivation spécifique du gène sla-1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-1 Download PDF

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WO2016197359A1
WO2016197359A1 PCT/CN2015/081231 CN2015081231W WO2016197359A1 WO 2016197359 A1 WO2016197359 A1 WO 2016197359A1 CN 2015081231 W CN2015081231 W CN 2015081231W WO 2016197359 A1 WO2016197359 A1 WO 2016197359A1
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sla
gene
sequence
sgrna
target sequence
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PCT/CN2015/081231
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Chinese (zh)
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蔡志明
牟丽莎
陈鹏飞
谢崇伟
张军方
高汉超
陆赢
刘璐
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深圳市第二人民医院
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
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    • 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
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12N2810/00—Vectors comprising a targeting moiety
    • C12N2810/10—Vectors comprising a non-peptidic targeting moiety

Definitions

  • the invention relates to the field of genetic engineering technology, in particular to the field of gene knockout technology, and particularly relates to a method for specifically knocking out the pig SLA-1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the SLA-1 gene.
  • Organ transplantation is the most effective treatment for organ failure diseases. To date, nearly one million patients worldwide have survived through organ transplantation. With the aging of the population and advances in medical technology, more and more patients need organ transplant surgery, but the shortage of donor organs severely restricts the development of organ transplant surgery. Taking kidney transplantation as an example, there are as many as 300,000 patients who need kidney transplantation every year in China, and no more than 10,000 donated kidneys for transplantation. Most of the patients die from kidney failure. Relying on post-mortem organ donation can no longer meet the needs of organ transplantation. Genetic engineering of other species to provide organs suitable for human transplantation has become the main way to address the shortage of human donor organs.
  • Primate T cells recognize pig-derived antigens and cause immune rejection against xenogeneic.
  • the primate T cell receptor recognizes the SLA/peptide complex on the surface of the porcine cell and directly activates the primate T cell.
  • there are two main types of donor-derived cell types that activate primate T cells one is a migratory antigen-presenting cell such as a dendritic cell; the other is a vascular endothelial cell that highly expresses SLA. .
  • MHC Class I molecules expressed on the surface of pig donor cells recognize and activate CD8T+ cells.
  • MHC class I molecules are glycoproteins composed of two peptide chains joined by non-covalent bonds; one of them is called a heavy chain or an alpha chain, and the other is a light chain or called ⁇ 2 microglobulin ( ⁇ 2m).
  • the molecular weight of the ⁇ chain is 44 kd, and the structure is polymorphic.
  • the extracellular domain peptide of the ⁇ chain is folded to form three functional regions, which are called ⁇ 1, ⁇ 2, and ⁇ 3 regions; each functional region contains about 90 amino acid residues, and its structure is similar to immunoglobulin (Ig); ⁇ 1
  • the amino acid sequence of the ⁇ 2 region varies greatly, which determines the polymorphism of the class I molecule.
  • 22m is not a MHC gene encoding, but a product encoded by a single gene on chromosome 15, with a molecular weight of 12kd. Its structure has greater homology with the ig constant region (ch3), belonging to Ig A member of the super family, without a homotypic determinant, but of species specificity.
  • the important physiological function of class I molecules is to limit the antigen recognition function of CD8+ T cells, that is, to participate in the process of presenting antigens to CD8+ T cells.
  • CD8+ T cells can only recognize antigens that bind to the same class I molecule (mostly endogenous cellular antigens, such as virus-infected cells and tumor cells, etc.), a phenomenon known as MHC restriction.
  • NK cells In addition to CD8+ T cells, NK cells also require the involvement of MHC class I molecules when they recognize that target cells exert a killing function. Therefore, if knockout MHC class I molecules will be able to control the cell killing effect of NK cells and CD8+ T cells, it will make an important contribution to xenotransplantation. The best way to achieve this strategy is to construct genetically modified pigs with missing MHC class I molecules.
  • common gene knockout techniques include homologous recombination (HR) technology, Transcription Activator-Like Effector Nuclease (TALEN) technology, Zinc-Finger Nuclease (ZFN) Technology and the recently developed Law Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) technique.
  • HR homologous recombination
  • TALEN Transcription Activator-Like Effector Nuclease
  • ZFN Zinc-Finger Nuclease
  • CRISPR Law Clustered Regularly Interspaced Short Palindromic Repeat Due to the inefficient recombination of HR technology (efficiency is only about 10 -6 ), the screening of mutants is very time consuming and inefficient, and has gradually been replaced.
  • the cutting efficiency of TALEN technology and ZFN technology can generally reach 20%, but all need to build protein modules that can recognize specific sequences, and the preliminary work is cumbersome and time consuming.
  • the module design of ZFN technology is complex and has a high off
  • CRISPR is an acquired immune system derived from prokaryotes that performs a function of interfering functions consisting of protein Cas and CRISPR-RNA (crRNA).
  • 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), to target sequences.
  • CRISPR RNA cryRNA
  • tracrRNA trans-activating crRNA
  • the two small RNAs have now been fused into an RNA strand, abbreviated as sgRNA (single guide RNA), which recognizes specific gene sequences and directs Cas9 protein for cleavage.
  • sgRNA single guide RNA
  • the CRISPR technology is simple in operation, high in screening efficiency, and capable of achieving accurate targeted cutting. Therefore, knocking out the SLA-1 gene by CRISPR technology can greatly improve the screening efficiency of SLA-1 deletion cells and genetically engineered pigs.
  • the key technical challenge of this path is to design and prepare precisely targeted sgRNAs, because the targeting accuracy of genes is highly dependent on sgRNA target sequences, and the successful design of precisely targeted sgRNAs becomes a key technical issue for knocking out target genes.
  • the present invention is intended to solve this technical problem and thereby provide a solid basis for knocking out the SLA-1 gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine SLA-1 gene and an sgRNA for specifically targeting the SLA-1 gene.
  • the present invention provides an sgRNA for specifically targeting an SLA-1 gene in a CRISPR-Cas9 specific knockout porcine SLA-1 gene, the sgRNA having the following characteristics:
  • the target sequence of the sgRNA on the SLA-1 gene conforms to the sequence arrangement rule of 5'-N(20)NGG-3', wherein N(20) represents 20 consecutive bases, wherein each N represents A Or T or C or G, a rule-compliant target sequence may be located in the sense strand or the antisense strand;
  • the target sequence of the sgRNA on the SLA-1 gene is located in the four exon coding regions at the N-terminus of the SLA-1 gene, or the major portion of the sequence is located in the four exons of the N-terminus of the SLA-1 gene, and the rest Partially crossing the boundary with adjacent introns, located adjacent to the intron;
  • the target sequence of the sgRNA on the SLA-1 gene is unique.
  • the above target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 162 in the Sequence Listing.
  • the above target sequence is the sequence shown by SEQ ID NO: 1 or 2 in the Sequence Listing.
  • the present invention provides a method for CRISPR-Cas9 specific knockout of a porcine SLA-1 gene, the method comprising the steps of:
  • the 5'-end of the target sequence of the sgRNA described in the first aspect is added to the sequence for forming the cohesive end, and the forward oligonucleotide sequence is synthesized; the target sequence of the sgRNA described in the first aspect
  • the opposite ends of the corresponding complementary sequences are added with appropriate sequences for forming sticky ends, and the reverse oligonucleotide sequence is synthesized; the synthesized forward oligonucleotide sequence is annealed to the reverse oligonucleotide sequence, To form a double-stranded oligonucleotide having a sticky end;
  • the above expression vector is a vector of the sequence shown by SEQ ID NO: 163 in the Sequence Listing.
  • the above method comprises the following steps:
  • a forward oligonucleotide sequence is synthesized by adding a CACCG sequence to the 5'-end of the target sequence of the sgRNA of the first aspect; the target sequence corresponding to the target sequence of the sgRNA of the first aspect is The 5'-end plus the AAAC sequence and the 3'-end plus C, the reverse oligonucleotide sequence is synthesized; the synthesized forward oligonucleotide sequence is annealed and renatured with the reverse oligonucleotide sequence, Forming a double-stranded oligonucleotide having a cohesive terminus;
  • the above double-stranded oligonucleotide is ligated into a linearized vector obtained by digesting the expression vector lentiCRISPR v2 of the sequence shown by SEQ ID NO: 163 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA.
  • the recombinant expression vector lentiCRISPR v2-SLA-1 of the oligonucleotide was transformed into competent bacteria, and the correct positive clone was screened, and the positive clone was shaken and the plasmid was extracted;
  • the above packaging plasmid is plasmid pLP1, plasmid pLP2 and plasmid pLP/VSVG; and the above packaging cell line is HEK293T cells.
  • the above target cells are porcine PIEC cells.
  • the gene fragment comprising the target sequence is amplified by using the genomic DNA as a template, and the knockdown of the SLA-1 gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the invention provides a CRISPR-Cas9 specific knockout pig SLA-1
  • the target sequence is preferably the target sequence shown by SEQ ID NO: 1 or 2 in the Sequence Listing.
  • the present invention provides a method for specifically knocking out a porcine SLA-1 gene by using the sgRNA according to the first aspect or the recombinant expression vector lentiCRISPR v2-SLA-1 of the third aspect in CRISPR-Cas9 Use in.
  • the present invention specifically knocks out the porcine SLA-1 gene for CRISPR-Cas9, successfully finds sgRNA that specifically targets the SLA-1 gene, and uses the sgRNA of the present invention for the CRISPR-Cas9 specific knockout porcine SLA-1 gene
  • the pig SLA-1 gene can be knocked out quickly, accurately, efficiently and specifically, and the technical problem of constructing the SLA-1 gene knockout pig with long cycle and high cost is effectively solved.
  • Figure 1 is a plasmid map of the vector plasmid lentiCRISPR v2 used in the examples of the present invention
  • Figure 2 is a plasmid map of the packaging plasmid pLP1 used in the embodiment of the present invention
  • Figure 3 is a plasmid map of the packaging plasmid pLP2 used in the examples of the present invention.
  • Figure 4 is a plasmid map of the packaging plasmid pLP/VSVG used in the examples of the present invention.
  • FIG. 5 is a diagram showing the results of electrophoresis detection of the gene knockout effect of the target sequence of the enzyme digestion in the embodiment of the present invention, wherein M represents DNA Marker, and Ctrl represents a targeted cutting effect of a control sequence which cannot effectively target the SLA-1 gene, 1 And 2 indicate the targeted cleavage effect of the No. 1 and No. 2 target sequences in Table 1 on the SLA-1 gene, respectively, and the arrow indicates a small fragment obtained by cutting with a Cruiser enzyme.
  • test materials and reagents involved in the following examples lentiCRISPR v2 plasmid was purchased from Addgene, packaging plasmids pLP1, pLP2 and pLP/VSVG were purchased from Invitrogen, and packaging cell line HEK293T cells were purchased from the American Model Culture Collection (ATCC).
  • PIEC cells were purchased from the Chinese Academy of Sciences cell bank, DMEM medium, Opti-MEM medium and fetal bovine serum FBS were purchased from Gibco, and Lipofectamine 2000 was purchased from Invitrogen.
  • a suitable 20 bp oligonucleotide sequence was searched for as a target sequence in the exon region of the SLA-1 gene.
  • the above target sequence and complementary sequence are separately added to the linker to form a forward oligonucleotide sequence and a reverse oligonucleotide sequence.
  • the above double-stranded DNA fragment was constructed into a vector of interest (e.g., lenti CRISPR V2, the plasmid map of which is shown in Figure 1) to form a lentiviral CRISPR vector such as lenti CRISPR SP2-SLA-1.
  • a vector of interest e.g., lenti CRISPR V2, the plasmid map of which is shown in Figure 1
  • lentiviral CRISPR vector such as lenti CRISPR SP2-SLA-1.
  • a CRISPR pseudotyped lentivirus expressing SLA-1sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.
  • a pseudotype lentivirus such as lenti CRISPR SP2-SLA-1 is added to the cell culture medium of interest for infection and further culture.
  • the target cells are collected, and the gene fragment containing the target sequence is amplified by using genomic DNA as a template, and the knockdown of the SLA-1 gene is determined by denaturation, renaturation and restriction enzyme digestion.
  • a number of single cell derived cell lines are isolated by dilution and monoclonal culture.
  • the target sequence determines the targeting specificity of the sgRNA and the efficiency of the Cas9-cleaving gene of interest. Therefore, efficient and specific target sequence selection and design are prerequisites for the construction of sgRNA expression vectors.
  • N(20) represents 20 contiguous bases, wherein each N represents A Or T or C or G, a rule-compliant target sequence may be located in the sense strand or the antisense strand;
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence can be complementary to form a double-stranded DNA fragment having a sticky end:
  • Example 2 sgRNA expression vector for constructing SLA-1 gene
  • Oligonucleotide sequences can be specifically synthesized by commercial companies (such as Invitrogen) according to the sequences provided. This example and the following examples investigate the knockdown effect of the target sequence shown by the sequences No. 1 and No. 2 listed in Table 1 on the SLA-1 gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the No. 1 target sequence are as follows:
  • AAACGGCGGGTCCCCACTCCCTGAC (SEQ ID NO: 165).
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 2 are as follows:
  • AAACAGGCGGGTCCCCACTCCCTGC (SEQ ID NO: 167).
  • the corresponding forward and reverse oligonucleotide sequences are annealed and renatured to form a double-stranded DNA fragment having sticky ends.
  • the reaction system (20 ⁇ L) is as follows:
  • the above reaction system was placed in a PCR machine, and the reaction was carried out in accordance with the following procedure.
  • the target vector lentiCRISPR v2 plasmid (the sequence of which is shown in SEQ ID NO: 163 in the Sequence Listing) was digested with BsmB I restriction endonuclease.
  • the digestion reaction system was placed at 37 ° C for 4 h.
  • the digestion mixture was separated by agarose gel electrophoresis, and the vector fragment (about 12 kb) was selected for cleavage and recovered by a DNA gel recovery column.
  • the double-stranded DNA fragment obtained by renaturation is linked with the recovered vector fragment, and is prepared according to the following reaction system:
  • Double-stranded DNA fragment 200ng
  • the ligation mixture was reacted at 25 ° C for 2 h.
  • the ligation mixture was transformed into E. coli DH5 ⁇ strain: 100 ⁇ L of E. coli DH5 ⁇ competent cells were added to the ligation mixture, and incubated on ice for 30 min; the mixture was placed in a 42 ° C water bath, heat shocked for 90 s, and then placed on ice to cool; 100 ⁇ L of LB medium was added and incubated at 37 ° C for 20 min on a shaker; the mixture was coated with Amp LB plates and incubated at 37 ° C for 14 h.
  • Example 3 obtaining a pseudotype lentivirus expressing SLA-1sgRNA
  • Amplify and extract the packaging plasmids pLP1, pLP2 and pLP/VSVG (purchased from Invitrogen, the maps are shown in Figure 2, Figure 3 and Figure 4, respectively); amplify and extract the vector plasmid lentiCRISPR v2-SLA-1; culture Packaging cell line HEK293T cells (purchased from ATCC); DMEM medium, Opti-MEM medium and fetal bovine serum FBS (purchased from Gibco); Lipofectamine 2000 (purchased from Invitrogen); HEK293T cells cultured at 37 ° C with 5% CO 2 In the culture environment, the medium was DMEM medium containing 10% FBS.
  • Formulation of Mixture 1 comprising:
  • Opti-MEM 500 ⁇ L.
  • Formulation of Mixture 2 comprising:
  • Opti-MEM 500 ⁇ L.
  • mixture 1 and mixture 2 were mixed to form a transfection mixture and allowed to stand for 20 min.
  • the HEK293T medium was changed to serum-free DMEM medium, and the transfection mixture was added. After incubation at 37 ° C for 8 hours, the cells were replaced with 20% FBS DMEM medium, and the culture was continued.
  • Example 4 infecting the target cell and detecting the knockout effect of the target sequence
  • PIEC porcine hip arterial endothelial cells
  • DMEM medium and fetal bovine serum FBS purchased from Gibco
  • lentiCRISPR v2-SLA- of different target sequences sequence 1 and sequence 2 1 pseudotype lentivirus
  • PIEC cells were cultured in a 37 ° C culture environment containing 5% CO 2 in DMEM medium containing 10% FBS.
  • Day 1 Passage cells of interest to 6-well plates at approximately 20% fusion density. Each virus requires a 6-well and requires an efficiency of 6 wells.
  • Uninfected efficacious control cells should all be apoptotic (>95%) under the action of puromycin.
  • the infection efficiency of cells can be determined, and the infection efficiency of 90% or more can be achieved (apoptosis rate ⁇ 10%). If necessary, the virus supernatant can be concentrated or diluted to be infected to achieve appropriate infection efficiency.
  • GCGCCACTGCGGTTCCCGGTTAT (SEQ ID NO: 168);
  • GAGGGTGAGACACGACCCTC (SEQ ID NO: 169).
  • the amplified fragment of interest consists of a sgRNA target sequence with a size of 450 bp.
  • the position of the target sequence to both ends of the fragment is not less than 100 bp.
  • the amplification reaction system (20 ⁇ L) was as follows:
  • the above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.
  • the second to fourth steps are repeated for 35 cycles.
  • the purified DNA fragments are separately denatured and renatured to form hybrid DNA molecules (including mutant samples and wild-type samples).
  • the reaction system is as follows:
  • Genomic PCR fragment 200ng
  • reaction buffer 2 ⁇ L
  • the reaction system has a total of 9 ⁇ L
  • the above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.
  • the digested DNA fragment was subjected to electrophoresis on a 2% agarose gel, 100 V, 25 min.
  • the cutting condition of the target fragment is determined, and the gene knocking effect of the target sequence is judged.
  • mutant DNA The cleavage recognition of mutant DNA is based on the principle that infected cells express sgRNA and Cas9. Genomic DNA, if sgRNA-mediated Cas9 protein-targeted cleavage, is introduced to introduce mutations near the cleavage site (wild-type becomes mutant). Since the wild type and the mutant sequence do not match at this position, the hybrid molecule in which the wild type DNA amplified by the template and the mutant DNA undergoes renaturation will generate a local loop structure. The latter can be recognized and cleaved by the Cruiser enzyme, resulting in the hybrid DNA molecule being cleaved into small fragments.
  • the partially infected cell population was passaged, and 100 single cells were transferred to a 10 cm dish for culture.
  • the annealed hybrid DNA was cleaved with a Cruiser enzyme and incubated at 45 ° C for 20 min.
  • 18 monoclonal clones were able to detect small fragments, indicating that knockout occurred, and the knockout efficiency was over 90%, indicating that the target sequence shown in Sequence 1 has a high targeted knockout SLA- The role of 1 gene.
  • the lentiCRISPR v2-SLA-1 pseudotyped lentivirus infection target cell based on the target sequence shown in SEQ ID NO: 2, 20 randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 17 of them were monoclonal. It can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach more than 85%, indicating that the target sequence shown in sequence 2 has a high target for knocking out the SLA-1 gene.

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Abstract

L'invention concerne un procédé d'inactivation d'un gène SLA-1 porcin utilisant la spécificité de CRISPR-Cas9, et un ARNsg utilisé pour cibler de façon spécifique le gène SLA-1. La séquence cible de l'ARNsg sur le gène SLA-1 est conforme aux règles de la séquence 5'-N(20)NGG-3 ', N(20) représentant 20 bases consécutives, et chaque N représentant A ou T ou C ou G ; la séquence cible dans le gène SLA-1 est située au niveau des régions codant pour les 4 exons, ou à la jonction avec les introns adjacents, au niveau de l'extrémité N-terminale du gène SLA-1 ; la séquence cible dans le gène SLA-1 est unique.
PCT/CN2015/081231 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène sla-1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-1 WO2016197359A1 (fr)

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CN201580000476.8A CN105593367A (zh) 2015-06-11 2015-06-11 CRISPR-Cas9特异性敲除猪SLA-1基因的方法及用于特异性靶向SLA-1基因的sgRNA
PCT/CN2015/081231 WO2016197359A1 (fr) 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène sla-1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-1

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