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WO2016197361A1 - Procédé d'inactivation spécifique du gène ggta1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène ggta1 - Google Patents

Procédé d'inactivation spécifique du gène ggta1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène ggta1 Download PDF

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WO2016197361A1
WO2016197361A1 PCT/CN2015/081233 CN2015081233W WO2016197361A1 WO 2016197361 A1 WO2016197361 A1 WO 2016197361A1 CN 2015081233 W CN2015081233 W CN 2015081233W WO 2016197361 A1 WO2016197361 A1 WO 2016197361A1
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ggta1
gene
sgrna
ggta1 gene
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Chinese (zh)
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蔡志明
牟丽莎
谢崇伟
张军方
陆赢
刘璐
高汉超
陈鹏飞
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深圳市第二人民医院
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    • C12N15/1137—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 enzymes
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    • 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
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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, in particular to a method for specifically knocking out the porcine GGTA1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the GGTA1 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.
  • ⁇ -1,3-galactose compound ⁇ -1,3-Gal
  • GGTA1 alpha-galactosyltransferase 1
  • Human cells cannot synthesize ⁇ -1,3-galactose compounds due to the lack of a functional GGTA1 gene.
  • Such compounds are widely present in bacteria and non-primates.
  • the human body produces a large amount of antibodies against ⁇ -1,3-galactose compounds under the stimulation of bacteria in the body. If a pig's organ is transplanted to a human, an acute hyperreactivity reaction occurs due to the corresponding antibody, resulting in a graft failure.
  • 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 GGTA1 gene by CRISPR technology can greatly improve the screening efficiency of ⁇ -1,3-Gal-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 basis for knocking out the GGTA1 gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine GGTA1 gene and an sgRNA for specifically targeting the GGTA1 gene.
  • the present invention provides an sgRNA for specifically targeting a GGTA1 gene in a CRISPR-Cas9 specific knockout porcine GGTA1 gene, the sgRNA having the following characteristics:
  • the target sequence of the sgRNA on the GGTA1 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 GGTA1 gene is located in the 5 exon coding regions at the N-terminus of the GGTA1 gene, or a part of the target sequence is located at 5 N-terminal exons of the GGTA1 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 GGTA1 gene is unique.
  • the above target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 38 in the Sequence Listing.
  • the above target sequence is the sequence shown by SEQ ID NO: 1 in the Sequence Listing.
  • the present invention provides a method of specifically knocking out a porcine GGTA1 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: 39 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: 39 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA.
  • the recombinant expression vector lentiCRISPR v2-GGTA1 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 knockout of the GGTA1 gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the present invention provides a recombinant expression vector lentiCRISPR v2-GGTA1 for use in a method for CRISPR-Cas9 specific knockout of a porcine GGTA1 gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 39; 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-GGTA1 of the third aspect, in the method of CRISPR-Cas9 specific knockout of the porcine GGTA1 gene.
  • the invention specifically targets the CRISPR-Cas9 knock-out porcine GGTA1 gene, and successfully finds the sgRNA which specifically targets the GGTA1 gene, and uses the sgRNA of the present invention in the method of CRISPR-Cas9 specific knockout of the porcine GGTA1 gene, which can be rapidly
  • the GGTA1 gene was knocked out accurately, efficiently and specifically, and the technical problem of constructing GGTA1 knockout pigs with long cycle and high cost was 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, Ctrl represents a targeted cutting effect of a control sequence which cannot effectively target the GGTA1 gene, and 1 represents a table.
  • M represents DNA Marker
  • Ctrl represents a targeted cutting effect of a control sequence which cannot effectively target the GGTA1 gene
  • 1 represents a table.
  • 3 indicates the targeted cleavage effect of the No. 3 target sequence in Table 1 on the GGTA1 gene
  • WT indicates the PCR of wild-type cells not infected with the virus and Cas9.
  • the result of the product Cruisein digestion test, the arrow indicates the small fragment obtained by cutting with the Cruiser enzyme;
  • FIG. 6 is a diagram showing the results of electrophoretic detection of the GGTA1 gene targeted cleavage effect of six PIEC cell monoclonals based on sequence 1 in the embodiment of the present invention, wherein the arrow indicates a small fragment obtained by cutting with a Cruiser enzyme, wherein In addition to the weak, cells 1, 2, 3, 4, and 6 have obvious small fragments;
  • Figure 7 is a graph showing the results of sequencing identification of cells No. 1, 3 and 6 in Figure 6, showing that Sequence 1 efficiently produced a mutation in the GGTA1 gene, in which the sequencing result of No. 1 cell (Fig. 7A) was analyzed to include an insertion mutation (Fig. 7) 1 sequence in 7D); sequencing result of cell No. 3 (Fig. 7B), including deletion mutation (3 sequence in Fig. 7D); sequencing result of cell No. 6 (Fig. 7C), analysis containing deletion mutation (Fig. 7) The 6 sequences in 7D); the arrows in Figures 7A, B indicate that Sequence 1 mediates the theoretical cleavage site of Cas9 on the GGTA1 gene, and WT represents the wild type sequence.
  • 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.
  • GGTA1 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 GGTA1 gene.
  • GGTA1 gene sgRNA target sequence design
  • 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-GGTA1.
  • 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-GGTA1.
  • a CRISPR pseudotyped lentivirus expressing GGTA1sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.
  • a pseudotype lentivirus such as lentiCRISPR v2-GGTA1 was added to the cell culture medium of interest for infection and further culture.
  • the target cell is collected, and the gene fragment containing the target sequence is amplified by using genomic DNA as a template, and the knockout of the GGTA1 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.
  • Example 1 Selection and design of sgRNA target sequence of Sus scrofa (pig) GGTA1 gene
  • 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.
  • GGTA1 For the GGTA1 gene, the following principles should be followed in the selection of target sequences:
  • 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 GGTA1 gene, or a part of the target sequence is located at five N-terminal exons of the GGTA1 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 GGTA1 gene, and the residual truncated sequence does not form a functional protein;
  • the target sequence is unique on the GGTA1 gene.
  • GGTA1 gene sgRNA target sequence design
  • 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 GGTA1 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 sequence No. 1 listed in Table 1 on the GGTA1 gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the No. 1 target sequence are as follows:
  • AAACTTACAGTTGAGACAAGCAGCC (SEQ ID NO: 41).
  • 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: 39 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 GGTA1sgRNA
  • 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-GGTA1; 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-GGTA1 false for different target sequences (sequence 1 and control sequences) Type lentivirus
  • Type lentivirus 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.
  • the amplified fragment of interest contains the sgRNA target sequence and is 362 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: 44 GAATACATCAACAGCCCAGA in the Sequence Listing) was not effective in targeting the GGTA1 gene to produce cleavage, and thus no small fragment was detected; PCR product of wild-type cells (WT) not infected with virus No small fragments were detected; no significant cleavage bands were detected in SEQ ID NO:3, which may be difficult to observe due to inability to effectively target genomic DNA, or because of the low efficiency of targeted cleavage.
  • WT wild-type cells
  • Another possible reason is that if the PIEC cells used in the examples are polymorphic at this position and do not conform to the standard sequence, the sequence 3 will not be effectively targeted.
  • Sequence 1 was able to effectively target the GGTA1 gene to produce a cleavage, the presence of a small fragment was detected, indicating that Sequence 1 can specifically knock out the target sequence of the porcine GGTA1 gene as CRISPR-Cas9.
  • the partially infected cell population was passaged, and 100 single cells were transferred to a 10 cm dish for culture.
  • the GGTA1 gene fragment of the monoclonal and wild-type cells is amplified according to the aforementioned method, and the amplified gene fragment contains the sgRNA target sequence.
  • 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 monoclonal knockdown of the GGTA1 gene was identified (Fig. 7).
  • the results shown in Figure 6 show that the lentiCRISPR v2-GGTA1 pseudotyped lentivirus infection target cell based on the target sequence shown in SEQ ID NO: 1 was randomly selected from 100 single cells and assayed by Cruiser enzyme electrophoresis. Among them, all 6 monoclonals (the weaker monoclonal band 5) can detect small fragments, indicating that gene knockout occurs, and the knockout efficiency can reach 100%, indicating that the target sequence shown in sequence 1 is very high. The role of targeted knockout of the GGTA1 gene.
  • Figure 7 shows further sequencing of PCR fragments of single cell clones No. 1, 3 and 6 in Figure 6, to determine mutations near the cleavage site, wherein Figure 7A shows the sequencing results of the PCR fragment of single cell clone No. 1.
  • the assay contains an insertional mutation (1 sequence in Figure 7D with the insertion of a G nucleotide). This indicates that a frameshift mutation has occurred, which can clearly indicate successful targeting of the knockout GGTA1 gene. In contrast to the wild type sequence, the inserted G was located at the theoretical cleavage site.
  • Figure 7B is a result of sequencing of a PCR fragment of single cell clone No. 3, which was analyzed to contain a deletion mutation (3 sequence in Figure 7D).
  • Figure 7C shows the sequencing results of the PCR fragment of single cell clone No. 6, which was analyzed to contain another deletion mutation (6 sequence in Figure 7D).
  • the theoretical cleavage site of single cell clone No. 6 has disappeared after repair.
  • Both of these monoclonal sequences showed a frameshift, indicating that the GGTA1 gene was targeted for knockout.
  • the presence of the above insertion and deletion mutations indicates that sequence 1 can efficiently mediate targeted knockdown of the GGTA1 gene.

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Abstract

L'invention concerne un procédé d'utilisation de la spécificité de CRISPR-Cas9 pour inactiver un gène GGTA1 porcin, et un ARNsg utilisé pour cibler de façon spécifique le gène GGTA1. La séquence cible de l'ARNsg pour le ciblage spécifique du gène GGTA1 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 GGTA1 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 GGTA1.
PCT/CN2015/081233 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène ggta1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène ggta1 WO2016197361A1 (fr)

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