WO2016197358A1 - Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2 - Google Patents

Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2 Download PDF

Info

Publication number
WO2016197358A1
WO2016197358A1 PCT/CN2015/081230 CN2015081230W WO2016197358A1 WO 2016197358 A1 WO2016197358 A1 WO 2016197358A1 CN 2015081230 W CN2015081230 W CN 2015081230W WO 2016197358 A1 WO2016197358 A1 WO 2016197358A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
gene
fgl2
sgrna
target sequence
Prior art date
Application number
PCT/CN2015/081230
Other languages
English (en)
Chinese (zh)
Inventor
蔡志明
牟丽莎
高汉超
谢崇伟
陆赢
刘璐
陈鹏飞
张军方
Original Assignee
深圳市第二人民医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳市第二人民医院 filed Critical 深圳市第二人民医院
Priority to CN201580000471.5A priority Critical patent/CN105518139B/zh
Priority to PCT/CN2015/081230 priority patent/WO2016197358A1/fr
Publication of WO2016197358A1 publication Critical patent/WO2016197358A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • 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, in particular to a method for specifically knocking out the pig FGL2 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the FGL2 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.
  • Fibrinogen-like protein 2 has a highly conserved fibrinogen domain that can be expressed either on the cell membrane or secreted outside the cell.
  • FGL2 on the membrane surface is a direct prothrombinase that is secreted into the cell.
  • the extra FGL2 has an immunomodulatory effect.
  • FGL2 promotes the conversion of thrombin to thrombin.
  • Cellulose deposition is a typical feature of AVR. Deletion of FGL2 inhibits cellulose deposition by antibody neutralizing or FGL2 knockout mice.
  • Thrombin is a potential immune activator that activates platelets and acts directly on vascular smooth muscle cells and vascular endothelial cells, and activation of vascular endothelial cells promotes thrombus formation.
  • the FGL2-deficient mice as donors did reduce the production of AVR. Since FGL2 has such an important role for AVR, building genetically modified pigs with FGL2 deletions will likely make an important contribution to xenotransplantation.
  • 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 FGL2 gene by CRISPR technology can greatly improve the screening efficiency of FGL2-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 FGL2 gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine FGL2 gene and an sgRNA for specifically targeting the FGL2 gene.
  • the present invention provides an sgRNA for specifically targeting a FGL2 gene in a CRISPR-Cas9 specific knockout porcine FGL2 gene, the sgRNA having the following characteristics:
  • the target sequence of the sgRNA on the FGL2 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 FGL2 gene is located in the exon coding region of the FGL2 gene;
  • the target sequence of the sgRNA on the FGL2 gene is unique.
  • the above target sequence is the sequence shown by any one of SEQ ID NOS: 1-78 in the Sequence Listing.
  • the above target sequence is the sequence shown by SEQ ID NO: 3 or 25 in the Sequence Listing.
  • the present invention provides a method for CRISPR-Cas9 specific knockout of a porcine FGL2 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: 79 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: 79 in the sequence listing by BsmB I restriction endonuclease to obtain a sgRNA.
  • the recombinant expression vector lentiCRISPR v2-FGL2 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 FGL2 gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the present invention provides a recombinant expression vector lentiCRISPR v2-FGL2 used in a method of CRISPR-Cas9 specific knockout of a porcine FGL2 gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 79; the target sequence to be carried, such as the target sequence of the sgRNA of the first aspect, preferably the target sequence shown by SEQ ID NO: 3 or 25 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-FGL2 of the third aspect, in the method of CRISPR-Cas9 specific knockout of the porcine FGL2 gene.
  • the present invention is directed to CRISPR-Cas9 specific knockout of the porcine FGL2 gene, and successfully finds an sgRNA that specifically targets the FGL2 gene, and the sgRNA of the present invention can be used in a method of CRISPR-Cas9 specific knockdown of the pig FGL2 gene, which can be rapidly
  • the accurate, efficient and specific knockout of the porcine FGL2 gene can effectively solve the technical problem of constructing the FGL2 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.
  • Figure 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 1 and 2 respectively represent the target sequence No. 3 and No. 25 of Table 1 for the FGL2 gene.
  • Targeted cleavage effect WT indicates the results of the PCR product of the wild-type cells that have not undergone viral infection and Cas9 cleavage, and the arrow shows the small fragment obtained by cutting with the 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 FGL2 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.
  • a target vector such as lenti CRISPR V2, the plasmid map of which Figure 1 shows a lentiviral CRISPR vector such as lenti CRISPR V2-FGL2.
  • a CRISPR pseudotyped lentivirus expressing FGL2 sgRNA was produced using a packaging plasmid, a packaging cell line, and a lentiviral CRISPR vector.
  • a pseudotype lentivirus such as lentiCRISPR v2-FGL2 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 FGL2 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.
  • 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 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 knockdown effect of the target sequence shown in the sequences No. 3 and No. 25 listed in Table 1 on the FGL2 gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 3 are as follows:
  • AAACGCCCGGTGAGACTAGAAAGCC (SEQ ID NO: 81).
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 25 are as follows:
  • AAACCCTCAATCCTGCCAAACTGCC (SEQ ID NO: 83).
  • 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: 79 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 FGL2 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-FGL2; 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-FGL2 false for different target sequences (sequence 3 and sequence 25) 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.
  • CAGGCGACCCTGAAGCCCGT (SEQ ID NO: 85).
  • Primers SEQ ID NO: 84-85 were used to detect sequence number 3
  • primers SEQ ID NO: 86-87 were used to detect sequence number 25.
  • 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 renatured hybrid DNA (including the mutant sample and the wild type sample) was cleaved with a Cruiser enzyme, and 1 ⁇ L of the Cruiser enzyme was added to the denatured and renatured reaction mixture, and incubated at 45 ° C for 20 min.
  • 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.
  • sequence 3 and sequence 25 were able to effectively target the FGL2 gene to produce a cleavage, and thus the presence of a small fragment was detected, indicating that the sequence 3 And sequence 25 is capable of specifically knocking out the target sequence of the porcine FGL2 gene as CRISPR-Cas9.
  • the partially infected cell population was passaged, and 100 single cells were transferred to a 10 cm dish for culture.
  • a FGL2 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.
  • the lentiCRISPR v2-FGL2 pseudotyped lentivirus infection target cell based on the target sequence shown in SEQ ID NO: 25, 20 monoclonal clones randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 18 of them were detected. By cutting small fragments, it indicates that gene knockout occurs, and the knockout efficiency can reach more than 90%, indicating that the target sequence shown in sequence 25 has a high target for knocking out the FGL2 gene.

Abstract

L'invention concerne un procédé d'inactivation d'un gène FGL-2 utilisant la spécificité de CRISPR-Cas9, et un ARNsg utilisé pour cibler de façon spécifique le gène FGL-2. La séquence cible de l'ARNsg pour le ciblage spécifique du gène FGL-2 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 FGL-2 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 FGL-2.
PCT/CN2015/081230 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2 WO2016197358A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201580000471.5A CN105518139B (zh) 2015-06-11 2015-06-11 CRISPR-Cas9特异性敲除猪FGL2基因的方法及用于特异性靶向FGL2基因的sgRNA
PCT/CN2015/081230 WO2016197358A1 (fr) 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2015/081230 WO2016197358A1 (fr) 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2

Publications (1)

Publication Number Publication Date
WO2016197358A1 true WO2016197358A1 (fr) 2016-12-15

Family

ID=55724976

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2015/081230 WO2016197358A1 (fr) 2015-06-11 2015-06-11 Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2

Country Status (2)

Country Link
CN (1) CN105518139B (fr)
WO (1) WO2016197358A1 (fr)

Cited By (28)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
CN109963944A (zh) * 2017-06-20 2019-07-02 江苏恒瑞医药股份有限公司 体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
CN113151274A (zh) * 2021-04-15 2021-07-23 贵州省畜牧兽医研究所 一种调控猪脂肪沉积性能的新LncRNA应用、挖掘及鉴定方法
CN113564165A (zh) * 2021-07-19 2021-10-29 武汉市工程科学技术研究院 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
CN114350615A (zh) * 2021-12-20 2022-04-15 北京镁伽科技有限公司 Stat2基因缺失细胞株及其制备方法和应用
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11898270B2 (en) * 2017-07-03 2024-02-13 Huazhong Agricultural University Pig genome-wide specific sgRNA library, preparation method therefor and application thereof
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
CN111100876A (zh) * 2018-10-25 2020-05-05 立沃生物科技（深圳）有限公司 CRISPR-Cas9特异性敲除FAH基因的方法及特异性sgRNA
CN110438128B (zh) * 2019-08-20 2023-04-25 山西农业大学 利用CRISPR/Cas9系统敲除猪CCAR1基因的方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140186843A1 (en) * 2012-12-12 2014-07-03 Massachusetts Institute Of Technology Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
WO2014165573A1 (fr) * 2013-04-02 2014-10-09 University Of Connecticut Régulation du rejet du greffon de tissus et organes de donneurs et dérivés de cellules souches embryonnaires

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0975361B1 (fr) * 1997-05-15 2006-09-20 Trillium Therapeutics Inc. Methodes de modulation de la coagulation immune
CN102085378B (zh) * 2010-12-29 2013-08-21 华中科技大学同济医学院附属同济医院 hfgl2抑制剂在制备治疗肝癌的药物中的应用
CN104480144B (zh) * 2014-12-12 2017-04-12 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

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140186843A1 (en) * 2012-12-12 2014-07-03 Massachusetts Institute Of Technology Methods, systems, and apparatus for identifying target sequences for cas enzymes or crispr-cas systems for target sequences and conveying results thereof
WO2014165573A1 (fr) * 2013-04-02 2014-10-09 University Of Connecticut Régulation du rejet du greffon de tissus et organes de donneurs et dérivés de cellules souches embryonnaires

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank [O] NCBI; 25 August 2012 (2012-08-25), XP055337407, Database accession no. NM_001005152.2 *

Cited By (47)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10323236B2 (en) 2011-07-22 2019-06-18 President And Fellows Of Harvard College Evaluation and improvement of nuclease cleavage specificity
US10954548B2 (en) 2013-08-09 2021-03-23 President And Fellows Of Harvard College Nuclease profiling system
US11920181B2 (en) 2013-08-09 2024-03-05 President And Fellows Of Harvard College Nuclease profiling system
US10508298B2 (en) 2013-08-09 2019-12-17 President And Fellows Of Harvard College Methods for identifying a target site of a CAS9 nuclease
US11046948B2 (en) 2013-08-22 2021-06-29 President And Fellows Of Harvard College Engineered transcription activator-like effector (TALE) domains and uses thereof
US9999671B2 (en) 2013-09-06 2018-06-19 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US11299755B2 (en) 2013-09-06 2022-04-12 President And Fellows Of Harvard College Switchable CAS9 nucleases and uses thereof
US10912833B2 (en) 2013-09-06 2021-02-09 President And Fellows Of Harvard College Delivery of negatively charged proteins using cationic lipids
US10597679B2 (en) 2013-09-06 2020-03-24 President And Fellows Of Harvard College Switchable Cas9 nucleases and uses thereof
US10682410B2 (en) 2013-09-06 2020-06-16 President And Fellows Of Harvard College Delivery system for functional nucleases
US10858639B2 (en) 2013-09-06 2020-12-08 President And Fellows Of Harvard College CAS9 variants and uses thereof
US10465176B2 (en) 2013-12-12 2019-11-05 President And Fellows Of Harvard College Cas variants for gene editing
US11053481B2 (en) 2013-12-12 2021-07-06 President And Fellows Of Harvard College Fusions of Cas9 domains and nucleic acid-editing domains
US11124782B2 (en) 2013-12-12 2021-09-21 President And Fellows Of Harvard College Cas variants for gene editing
US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11578343B2 (en) 2014-07-30 2023-02-14 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US10704062B2 (en) 2014-07-30 2020-07-07 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
US11214780B2 (en) 2015-10-23 2022-01-04 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US10167457B2 (en) 2015-10-23 2019-01-01 President And Fellows Of Harvard College Nucleobase editors and uses thereof
US11702651B2 (en) 2016-08-03 2023-07-18 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10113163B2 (en) 2016-08-03 2018-10-30 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US10947530B2 (en) 2016-08-03 2021-03-16 President And Fellows Of Harvard College Adenosine nucleobase editors and uses thereof
US11661590B2 (en) 2016-08-09 2023-05-30 President And Fellows Of Harvard College Programmable CAS9-recombinase fusion proteins and uses thereof
US11542509B2 (en) 2016-08-24 2023-01-03 President And Fellows Of Harvard College Incorporation of unnatural amino acids into proteins using base editing
US11306324B2 (en) 2016-10-14 2022-04-19 President And Fellows Of Harvard College AAV delivery of nucleobase editors
US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
US11820969B2 (en) 2016-12-23 2023-11-21 President And Fellows Of Harvard College Editing of CCR2 receptor gene to protect against HIV infection
US11898179B2 (en) 2017-03-09 2024-02-13 President And Fellows Of Harvard College Suppression of pain by gene editing
US11542496B2 (en) 2017-03-10 2023-01-03 President And Fellows Of Harvard College Cytosine to guanine base editor
US11268082B2 (en) 2017-03-23 2022-03-08 President And Fellows Of Harvard College Nucleobase editors comprising nucleic acid programmable DNA binding proteins
US11560566B2 (en) 2017-05-12 2023-01-24 President And Fellows Of Harvard College Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation
CN109963944A (zh) * 2017-06-20 2019-07-02 江苏恒瑞医药股份有限公司 体外敲除T细胞中靶基因的方法以及该方法中使用的crRNA
US11898270B2 (en) * 2017-07-03 2024-02-13 Huazhong Agricultural University Pig genome-wide specific sgRNA library, preparation method therefor and application thereof
US11732274B2 (en) 2017-07-28 2023-08-22 President And Fellows Of Harvard College Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE)
US11319532B2 (en) 2017-08-30 2022-05-03 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11932884B2 (en) 2017-08-30 2024-03-19 President And Fellows Of Harvard College High efficiency base editors comprising Gam
US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
US11795452B2 (en) 2019-03-19 2023-10-24 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11447770B1 (en) 2019-03-19 2022-09-20 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
US11643652B2 (en) 2019-03-19 2023-05-09 The Broad Institute, Inc. Methods and compositions for prime editing nucleotide sequences
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
CN113151274A (zh) * 2021-04-15 2021-07-23 贵州省畜牧兽医研究所 一种调控猪脂肪沉积性能的新LncRNA应用、挖掘及鉴定方法
CN113151274B (zh) * 2021-04-15 2024-01-19 贵州省畜牧兽医研究所 一种调控猪脂肪沉积性能的新LncRNA应用、挖掘及鉴定方法
CN113564165B (zh) * 2021-07-19 2023-06-27 武汉市工程科学技术研究院 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用
CN113564165A (zh) * 2021-07-19 2021-10-29 武汉市工程科学技术研究院 一种胞内编辑伪狂犬病毒关键基因的细胞株及其构建方法和应用
CN114350615A (zh) * 2021-12-20 2022-04-15 北京镁伽科技有限公司 Stat2基因缺失细胞株及其制备方法和应用
CN114350615B (zh) * 2021-12-20 2024-04-16 北京镁伽科技有限公司 Stat2基因缺失细胞株及其制备方法和应用

Also Published As

Publication number Publication date
CN105518139A (zh) 2016-04-20
CN105518139B (zh) 2021-02-02

Similar Documents

Publication Publication Date Title
WO2016197358A1 (fr) Procédé d'inactivation spécifique du gène fgl-2 porcin utilisant la spécificité de crispr-cas9, et arnsg ciblant de façon spécifique le gène fgl-2
WO2016197355A1 (fr) Procédé crispr-cas9 pour inactivation spécifique du gène sall1 porcin et arnsg pour utilisation dans le ciblage spécifique du gène sall1
WO2016197354A1 (fr) Procédé crispr-cas9 pour inactivation spécifique du gène pdx1 porcin et arnsg pour utilisation dans le ciblage spécifique du gène pdx1
WO2016197360A1 (fr) Procédé d'inactivation spécifique du gène gfra1 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène gfra1
WO2016197357A1 (fr) Procédé d'inactivation spécifique du gène sla-3 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-3
WO2016197362A1 (fr) 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
WO2016197356A1 (fr) Procédé d'inactivation du gène sla-2 porcin utilisant la spécificité de crispr-cas9, et arnsg utilisé pour cibler de façon spécifique le gène sla-2
WO2016197361A1 (fr) 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
WO2016187904A1 (fr) Procédé d'inactivation spécifique d'un gene cmah du porc au moyen de crispr-cas9 et d'arnsg qui cible spécifiquement un gène cmah
WO2016197359A1 (fr) 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
CN105518135B (zh) CRISPR-Cas9特异性敲除猪CMAH基因的方法及用于特异性靶向CMAH基因的sgRNA
KR102553518B1 (ko) Hiv 감염의 rna-가이드된 치료를 위한 방법 및 조성물
US20210207169A1 (en) Viral delivery of rna utilizing self-cleaving ribozymes and crispr-based applications thereof
CN104480144B (zh) 用于艾滋病基因治疗的CRISPR/Cas9重组慢病毒载体及其慢病毒
US10988777B2 (en) Method for inducing CCR5Δ32 deletion by using CRISPR-Cas9 genome editing technique
US20210047375A1 (en) Lentiviral-based vectors and related systems and methods for eukaryotic gene editing
JP7370702B2 (ja) タンパク質製造用の改善された真核細胞およびそれらの作製方法
Qu et al. Zinc finger nuclease: a new approach for excising HIV-1 proviral DNA from infected human T cells
US20220090144A1 (en) Characterization and inactivation of endogenous retroviruses in chinese hamster ovary cells
JP2023508400A (ja) 遺伝子発現を増強させる哺乳動物配列への標的組込み
US20220364072A1 (en) Fusion protein that improves gene editing efficiency and application thereof
Zhang et al. Interfering with retrotransposition by two types of CRISPR effectors: Cas12a and Cas13a
CN112979823B (zh) 一种用于治疗和/或预防β血红蛋白病的产品及融合蛋白
CN110257424B (zh) 一种定点编辑ccr5基因的方法
Rousseau Engineering Virus-Like Particles for the Delivery of Genome Editing Enzymes

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15894628

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15894628

Country of ref document: EP

Kind code of ref document: A1