WO2016197362A1 - Procédé d'inactivation spécifique du gène vwf porcin utilisant la spécificité de crispr-cas9, et arngsg utilisé pour cibler de façon spécifique le gène vwf - Google Patents

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

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WO2016197362A1
WO2016197362A1 PCT/CN2015/081234 CN2015081234W WO2016197362A1 WO 2016197362 A1 WO2016197362 A1 WO 2016197362A1 CN 2015081234 W CN2015081234 W CN 2015081234W WO 2016197362 A1 WO2016197362 A1 WO 2016197362A1
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sequence
vwf
sgrna
gene
vwf gene
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PCT/CN2015/081234
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Chinese (zh)
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蔡志明
牟丽莎
高汉超
谢崇伟
刘璐
陈鹏飞
张军方
陆赢
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深圳市第二人民医院
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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
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
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Definitions

  • the invention relates to the field of genetic engineering technology, in particular to the field of gene knockout technology, in particular to a method for specifically knocking out a swine vWF gene by CRISPR-Cas9 and an sgRNA for specifically targeting a vWF 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.
  • vWF von Willebrand factor
  • Alpha (1,3) Gal epitope and activates primate platelets.
  • vWF is a kind of glycoprotein with large molecular weight and adhesion function, which can mediate platelet adhesion and promote hemostasis of wounds, while defects of vWF will lead to hemorrhage of mucosa and skin.
  • the vWF-mediated protein-protein interaction plays an important regulatory role in the hemostasis process and thrombus formation.
  • vWF-deficient pig lungs can also effectively reduce the common coagulopathy during swine-pigment xenografts.
  • Xenografting with vWF-deficient pig lungs also significantly reduced endothelial cell activation.
  • knocking out swine vWF by genetic engineering technology is expected to reduce the xenograft disability syndrome in xenografts using defective vWF pig-derived organs, which will make an important contribution to xenotransplantation.
  • the best way to achieve this strategy is to construct genetically modified pigs with missing vWF 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 vWF gene by CRISPR technology can greatly improve the screening efficiency of vWF-deficient 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 foundation for knocking out the vWF gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockout of the swine vWF gene and an sgRNA for specifically targeting the vWF gene.
  • the invention provides a CRISPR-Cas9 specific knockout porcine vWF group Because of the sgRNA used to specifically target the vWF gene, the sgRNA has the following characteristics:
  • the target sequence of the sgRNA on the vWF gene conforms to the sequence alignment 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 vWF gene is located in the exon coding region of the vWF gene;
  • the target sequence of the sgRNA on the vWF gene is unique.
  • the above target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 58 in the Sequence Listing.
  • the above target sequence is the sequence shown by SEQ ID NO: 1 in the Sequence Listing.
  • the invention provides a method for CRISPR-Cas9 specific knockout of a swine vWF 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: 59 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 oligo with sticky ends Polynucleotide
  • 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: 59 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA.
  • the recombinant expression vector lentiCRISPR v2-vWF 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 vWF gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the present invention provides a recombinant expression vector lentiCRISPR v2-vWF for use in a method for CRISPR-Cas9 specific knockout of a swine vWF gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 59; the target sequence carried, such as the target sequence of the sgRNA of the first aspect, is preferably the target sequence shown by SEQ ID NO: 1 in the sequence listing.
  • the present invention provides the use of the sgRNA according to the first aspect or the recombinant expression vector lentiCRISPR v2-vWF according to the third aspect, in the method of CRISPR-Cas9 specific knockout of the swine vWF gene.
  • the present invention specifically knocks out the swine vWF gene against CRISPR-Cas9 and successfully finds a specific target
  • the sgRNA of the vWF gene, the sgRNA of the invention is used in the method of CRISPR-Cas9 specific knockout of the swine vWF gene, and the swine vWF gene can be rapidly, accurately, efficiently and specifically knocked out, and the vWF gene knocking is effectively solved.
  • the technical problems of long pig cycle and high cost are examples of the technical problems of long pig cycle and high cost.
  • 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 knock-out effect of the target sequence in the enzyme digestion assay according to the embodiment of the present invention, wherein M represents DNA Marker, and 1 represents the targeted cleavage effect of the No. 1 target sequence in Table 1 on the vWF gene, WT
  • M represents DNA Marker
  • 1 represents the targeted cleavage effect of the No. 1 target sequence in Table 1 on the vWF gene, WT
  • the result of the PCR product of the wild type cell which has not been subjected to viral infection and Cas9 cleavage is the result of a Bruiser digestion test, 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 vWF 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 target vector (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-vWF.
  • a target vector e.g., lenti CRISPR V2, the plasmid map of which is shown in Figure 1
  • lentiviral CRISPR vector such as lenti CRISPR SP2-vWF.
  • a CRISPR pseudotyped lentivirus expressing a vWF sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.
  • a pseudotyped lentivirus such as lentiCRISPR v2-vWF 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 vWF 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 selection is performed in the shared exon coding region, and the exon coding region sequence near the N-terminus is selected for the vWF gene to satisfy the condition;
  • GGCCCACTCTCTTGCCATCT 20 CACAAAGCCGTAGGCCTCGC twenty one GTTGCCGCTCCCATCAATTC twenty two CAGCGAGGCCTACGGCTTTG twenty three AATTCTGGCCACAAAGCCGT twenty four GTACCACACGCTGTCCAGCG 25 ACGCTGTCCAGCGAGGCCTA 26 CCCTTTGCTGGCATAGGGCG 27 TGTCAATGGCACCGTGCTGC 28 GATACTTCAACAAGACCTGC 29 GTCAATGGCACCGTGCTGCA 30 CATAGGGCGTGGAGATGCTT 31 CGGTGCCATTGACAAACACA 32 TGAAGTATCTGTCTGACAGC 33 TCAATGGCACCGTGCTGCAG 34 TACAGCCCTTTGCTGGCATA 35 TGCCAGCAAAGGGCTGTATC 36 GGAGATGTTGCACGAGATGC 37 AAAGTCGCCACAGAGCCCGC 38 GGCAAAGCTGTAGGGGTCCG 39 GAGATGTTGCACGAGATGCT 40 AGCTGTAGGGGTCCGAGGTC 41 AGGGCATGCT
  • the CACCG sequence was added to the 5'-end of the above N(20) target sequence to form a forward oligonucleotide sequence according to the characteristics of the lenti CRISPR SP2 plasmid:
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence can be complementary to form a double-stranded DNA fragment having a sticky end:
  • 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 knockout effect of the target sequence shown in the first sequence listed in Table 1 on the vWF gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the No. 1 target sequence are as follows:
  • AAACGTACTCCATGCCCGCGGGGCC (SEQ ID NO: 61).
  • 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: 59 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 vWF sgRNA
  • 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-vWF; culture packaging cells 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 in 37 ° C culture environment containing 5% CO 2 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
  • the target cell line was porcine hip arterial endothelial cells PIEC (purchased from the Chinese Academy of Sciences cell bank); DMEM medium and fetal bovine serum FBS (purchased from Gibco); target sequence (sequence 1) lentiCRISPR v2-vWF pseudotype lentivirus;
  • the 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.
  • ACACCAGCTCAATCCTGATC SEQ ID NO: 63.
  • 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.
  • a small fragment was not detected in the PCR product of the wild-type cell which was not infected with the virus; and the sequence 1 was able to effectively target the vWF gene to produce a cleavage, and thus the presence of a small fragment was detected, indicating that the sequence 1 can be used as a CRISPR -Cas9 specifically knocks out the target sequence of the swine vWF gene.
  • the partially infected cell population was passaged, and 100 single cells were transferred to a 10 cm dish for culture.
  • a vWF gene fragment of monoclonal and wild-type cells is amplified according to the aforementioned method, and the amplified gene fragment comprises an sgRNA target sequence.
  • the annealed hybrid DNA was cleaved with a Cruiser enzyme and incubated at 45 ° C for 20 min.

Abstract

L'invention concerne un procédé d'utilisation de la spécificité de CRISPR-Cas9 pour inactiver un gène vWF porcin, et un ARNsg utilisé pour cibler de façon spécifique le gène vWF. La séquence cible de l'ARNsg pour le ciblage spécifique du gène vWF est conforme aux règles de la séquence 5'-N(20)NGG-3', N(20) représentant 20 bases consécutives et N représentant A ou T ou C ou G ; la séquence cible dans le gène vWF est unique et est située au niveau des régions codant pour les 5 exons, ou à la jonction avec les introns adjacents, au niveau de l'extrémité N-terminale du gène vWF.
PCT/CN2015/081234 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène vwf porcin utilisant la spécificité de crispr-cas9, et arngsg utilisé pour cibler de façon spécifique le gène vwf WO2016197362A1 (fr)

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PCT/CN2015/081234 WO2016197362A1 (fr) 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène vwf porcin utilisant la spécificité de crispr-cas9, et arngsg utilisé pour cibler de façon spécifique le gène vwf

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US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106434752A (zh) * 2016-06-14 2017-02-22 南通大学附属医院 敲除Wnt3a基因的过程及其验证方法
CN106399373B (zh) * 2016-11-18 2019-05-24 青岛市畜牧兽医研究所 一种Cas9表达载体

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014071235A1 (fr) * 2012-11-01 2014-05-08 Massachusetts Institute Of Technology Dispositif génétique pour la destruction régulée d'adn
WO2014082644A1 (fr) * 2012-11-30 2014-06-05 WULFF, Peter, Samuel Arn circulaire destiné à l'inhibition de micro-arn
CN104480144A (zh) * 2014-12-12 2015-04-01 武汉大学 用于艾滋病基因治疗的CRISPR/Cas9重组慢病毒载体及其慢病毒

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL160799A0 (en) * 2001-09-12 2004-08-31 Virexx Medical Corp Vascular occlusion solid-phase agent with immobilised platelet binding agent

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014071235A1 (fr) * 2012-11-01 2014-05-08 Massachusetts Institute Of Technology Dispositif génétique pour la destruction régulée d'adn
WO2014082644A1 (fr) * 2012-11-30 2014-06-05 WULFF, Peter, Samuel Arn circulaire destiné à l'inhibition de micro-arn
CN104480144A (zh) * 2014-12-12 2015-04-01 武汉大学 用于艾滋病基因治疗的CRISPR/Cas9重组慢病毒载体及其慢病毒

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] NCBI; 2 June 2011 (2011-06-02), "TPA_inf: Sus scrofa von Willebrand factor (VWF) mRNA, exons 1 through", XP055337394, Database accession no. BK007995.1 *

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