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WO2016187904A1 - Procédé d'inactivation spécifique d'un gene cmah du porc au moyen de crispr-cas9 et d'arnsg qui cible spécifiquement un gène cmah - Google Patents

Procédé d'inactivation spécifique d'un gene cmah du porc au moyen de crispr-cas9 et d'arnsg qui cible spécifiquement un gène cmah Download PDF

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WO2016187904A1
WO2016187904A1 PCT/CN2015/081347 CN2015081347W WO2016187904A1 WO 2016187904 A1 WO2016187904 A1 WO 2016187904A1 CN 2015081347 W CN2015081347 W CN 2015081347W WO 2016187904 A1 WO2016187904 A1 WO 2016187904A1
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sequence
sgrna
cmah
cmah gene
gene
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蔡志明
牟丽莎
谢崇伟
高汉超
刘璐
陈鹏飞
张军方
陆赢
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Shenzhen Second Peoples Hospital
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    • 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
    • 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
    • C12N15/63—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
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors

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 a pig CMAH gene by CRISPR-Cas9 and an sgRNA for specifically targeting a CMAH 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.
  • CMAH cytidine monophosphate-N-acetylneuraminic acid hydroxylase
  • 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 time consuming and inefficient and has been gradually 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-target
  • 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 CMAH gene by CRISPR technology can greatly improve the screening efficiency of Neu5Gc-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 the technical problem and thereby provide a solid foundation for knocking out the CMAH gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine CMAH gene and an sgRNA for specifically targeting the CMAH gene.
  • the present invention provides an sgRNA for specifically targeting a CMAH gene in a CRISPR-Cas9 specific knockout porcine CMAH gene, the sgRNA having the following characteristics:
  • the target sequence of the sgRNA on the CMAH 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, the target sequence conforming to the above rules is located in the sense strand or the antisense strand;
  • the target sequence of the sgRNA on the CMAH gene is located in the 5 exon coding regions at the N-terminus of the CMAH gene, or a part of the target sequence is located at 5 N-terminal exons of the CMAH gene, and the remaining portions span and adjacent The junction of the inclusions, located in adjacent introns;
  • the target sequence of the sgRNA on the CMAH gene is unique.
  • the above target sequence is any one of SEQ ID NOS: 1 to 61 in the sequence listing. The sequence shown by the sequence of bars.
  • the above target sequence is the sequence shown by SEQ ID NO: 2 or 4 in the Sequence Listing.
  • the present invention provides a method of specifically knocking out a porcine CMAH gene using CRISPR-Cas9, 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: 62 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: 62 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA.
  • the recombinant expression vector lentiCRISPR v2-CMAH of the oligonucleotide was transformed into competent bacteria, and the correct positive clone was identified by screening, 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 CMAH gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the present invention provides a recombinant expression vector lentiCRISPR v2-CMAH used in a method for CRISPR-Cas9 specific knockout of a porcine CMAH gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 62; 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: 2 or 4 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-CMAH according to the third aspect, in the method of CRISPR-Cas9 specific knockout of the porcine CMAH gene.
  • the invention specifically targets the CRISPR-Cas9 knock-out porcine CMAH gene, and successfully finds the sgRNA which specifically targets the CMAH gene, and uses the sgRNA of the present invention in the method of CRISPR-Cas9 specific knockout of the porcine CMAH gene, which can be rapidly Accurate, efficient and specific knockout of the porcine CMAH gene, effectively solving the technical problems of constructing the CMAH gene knockout pig with long 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 knockout effect of the target sequence of the enzyme digestion in the embodiment of the present invention, wherein M represents DNA Marker, and Ctrl indicates a targeted cutting effect of a control sequence which cannot effectively target the CMAH gene, 2 and 4
  • M represents DNA Marker
  • Ctrl indicates a targeted cutting effect of a control sequence which cannot effectively target the CMAH gene
  • WT indicates the results of the PCR product of the wild-type cells not subjected to viral infection and Cas9 cleavage, and the results indicated by the arrow are shown by the Cruiser. a small fragment obtained by enzymatic cleavage;
  • FIG. 6 is a diagram showing the results of electrophoretic detection of the CMAH gene targeted cleavage effect of seven PIEC cell monoclonals based on sequence 2 in the embodiment of the present invention, wherein the arrow indicates a small fragment obtained by cutting with a Cruiser enzyme, wherein 1, 2, 3, Cells 4, 6, and 7 have obvious small fragments;
  • Figure 7 is a graph showing the results of sequencing identification of cells Nos. 3 and 7 in Figure 6, showing that Sequence 2 effectively produced a mutation in the CMAH gene, wherein the sequencing result of the No. 3 cell (Fig. 7A) was analyzed to include a deletion mutation (Fig. 7)
  • the B1 sequence in 7C), the clone also contains a part of the wild-type gene sequence (WT), which appears as a bimodal phenomenon in the latter half of Figure 7A, and the arrow in Figure 7A indicates that sequence 2 mediates the theoretical cleavage of Cas9 on the CMAH gene.
  • the sequencing results of the No. 7 cells (Fig.
  • 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 cell bank of the Chinese Academy of Sciences, DMEM medium, Opti-MEM medium and fetal bovine serum FBS were purchased from Gibco, and Lipofectamine 2000 was purchased from Invitrogen.
  • CMAH gene sgRNA target sequence selection 1.
  • a suitable 20 bp oligonucleotide sequence was searched for as a target sequence in the exon region of the CMAH 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-CMAH.
  • 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-CMAH.
  • a CRISPR pseudotyped lentivirus expressing CMAH sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.
  • a pseudotype lentivirus such as lentiCRISPR v2-CMAH is added to the cell culture medium of interest for infection and further culture.
  • the target cells were collected, and the gene fragment containing the target sequence was amplified by using genomic DNA as a template, and the knockdown of the CMAH gene was 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.
  • CMAH complementary metal-oxide-semiconductor
  • N(20) represents 20 contiguous bases, wherein each N represents A or T Or C or G, the target sequence conforming to the above rules is located in the sense strand or the antisense strand;
  • the target sequence may be located in the five exon coding regions at the N-terminus of the CMAH gene, or a part of the target sequence is located at five N-terminal exons of the CMAH gene, The remainder spans the junction with adjacent introns and is located adjacent to the intron; such cleavage of the coding region sequence results in functional knockdown of the CMAH gene, and the residual truncated sequence does not form a functional protein;
  • the target sequence is unique on the CMAH gene.
  • 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:
  • Example 2 Construction of sgRNA expression vector of CMAH 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 knockout effect of the target sequence shown in the sequences No. 2 and No. 4 listed in Table 1 on the CMAH gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 2 are as follows:
  • AAACGCCGAAGCTGCCAATCTCAAC (SEQ ID NO: 64).
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 4 are as follows:
  • AAACTGAGATTGGCAGCTTCGGCAC (SEQ ID NO: 66).
  • 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: 62 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; Add 100 ⁇ L of LB medium and incubate at 37 ° C for 20 min on a shaker; The mixture was coated on an Amp LB plate and incubated at 37 ° C for 14 h.
  • Example 3 obtaining a pseudotype lentivirus expressing CMAH 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-CMAH; 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
  • PIEC porcine hip arterial endothelial cells
  • DMEM medium and fetal bovine serum FBS purchased from Gibco
  • lentiCRISPR v2-CMAH fake of different target sequences (sequence 2 and sequence 4) Type 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.
  • CTTCCATTTAGATCCTCAATGTCTTTAATGAAG (SEQ ID NO: 68).
  • the amplified fragment of interest contains the sgRNA target sequence and is 460 bp in size.
  • 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 control sequence (SEQ ID NO: 69 in the Sequence Listing) could not effectively target the CMAH gene to produce a cleavage, and thus no small fragment was detected; the PCR product of the wild-type cell not infected with the virus was also not detected.
  • the small fragment While Sequence 2 and Sequence 4 were able to effectively target the CMAH gene to produce cleavage, the presence of a small fragment was detected, indicating that Sequence 2 and Sequence 4 can serve as CRISPR-Cas9 specific knockout target sequences for the porcine CMAH gene.
  • 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.
  • the monoclonal PCR fragment of the effective mutation is further sequenced to determine the mutation near the target sequence.
  • a single knockout of the CMAH gene was identified (Fig. 7).
  • the results shown in Figure 6 show that the lentiCRISPR v2-CMAH pseudotype lentivirus infection target cell based on the target sequence shown in SEQ ID NO: 2, 7 monoclonal clones randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis. Among them, 6 monoclonals (ie, monoclonals 1, 2, 3, 4, 6, and 7) can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach 80% or more, indicating that sequence 2 The indicated target sequence has a high target for knocking out the CMAH gene.
  • 6 monoclonals ie, monoclonals 1, 2, 3, 4, 6, and 7
  • 6 monoclonals can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach 80% or more, indicating that sequence 2
  • the indicated target sequence has a high target for knocking out the CMAH gene.
  • Figure 7 shows the further sequencing of the PCR fragments of the single cell clones No. 3 and No. 7 in Figure 6 to determine the mutation near the cleavage site
  • Figure 7A shows the sequencing results of the PCR fragment of the single cell clone No. 3, followed by
  • the half-section has a bimodal phenomenon, which is analyzed to contain a deletion mutation (B1 sequence in Figure 7C) and a partial wild type, indicating that a targeted knockout of the CMAH gene occurs, and the presence of the wild type may be two in the genome.
  • B1 sequence in Figure 7C a deletion mutation
  • a partial wild type indicating that a targeted knockout of the CMAH gene occurs, and the presence of the wild type may be two in the genome.
  • One of the alleles on the chromosome did not undergo targeted knockout or some wild-type cells did not mutate, however the presence of the deletion mutation clearly indicated successful targeting of the knockout of the CMAH gene.
  • Figure 7B shows the sequencing results of the PCR fragment of single cell clone No. 7.
  • the second half has a bimodal phenomenon, and the analysis includes two mutations: one is an insertion mutation (named D4-1 in Figure 7C), and the other is The deletion mutation (designated D4-2 in Figure 7C) indicates that the CMAH gene is targeted for knockout.
  • Two different types of mutations may have not been genetically repaired when the monoclonal was picked, and then the split cells They are repaired into different mutation types, or the alleles on both chromosomes are knocked out and repaired into different genotypes.
  • the presence of insertional and deletion mutations indicates a targeted knockout effect on the CMAH gene.

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Abstract

L'invention concerne un procédé d'inactivation spécifique d'un gène CMAH du porc au moyen de CRISPR-Cas9 et d'ARNsg qui cible spécifiquement un gène CMAH. Une séquence cible de l'ARNsg qui cible spécifiquement le gène CMAH respecte une règle d'arrangement de séquence en 5'-N(20)NGG-3', N(20) représentant 20 bases consécutives, et N représentant A ou T ou C ou G ; la séquence cible sur le gène CMAH est unique, et est située au niveau d'une région de codage de l'exon 5 d'un N terminal du gène CMAH ou au niveau d'une position de jonction avec les introns adjacents.
PCT/CN2015/081347 2015-05-22 2015-06-12 Procédé d'inactivation spécifique d'un gene cmah du porc au moyen de crispr-cas9 et d'arnsg qui cible spécifiquement un gène cmah Ceased WO2016187904A1 (fr)

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