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WO2016197360A1 - 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 - Google Patents

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 Download PDF

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WO2016197360A1
WO2016197360A1 PCT/CN2015/081232 CN2015081232W WO2016197360A1 WO 2016197360 A1 WO2016197360 A1 WO 2016197360A1 CN 2015081232 W CN2015081232 W CN 2015081232W WO 2016197360 A1 WO2016197360 A1 WO 2016197360A1
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sequence
gfra1
gene
sgrna
gfra1 gene
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PCT/CN2015/081232
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Chinese (zh)
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蔡志明
牟丽莎
谢崇伟
刘璐
陈鹏飞
张军方
陆赢
高汉超
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深圳市第二人民医院
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Priority to CN201580000470.0A priority Critical patent/CN105518138B/zh
Priority to PCT/CN2015/081232 priority patent/WO2016197360A1/fr
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    • 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
    • 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
    • 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 porcine GFRA1 gene by CRISPR-Cas9 and an sgRNA for specifically targeting the GFRA1 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.
  • the traditional technical route achieves a strain of pigs that can be used for transplantation by reducing the difference in immunity between pigs and humans.
  • organ-deficient pigs as a culture environment to produce organs composed of human cells has become a new idea. Through genetic engineering, it effectively interferes with the genes that control the development of pig's own organs, so that an organ is missing during development, which provides a key culture environment for the development of human cell organs.
  • the GFRA1 gene is currently known to be an essential gene in kidney development.
  • GFRA1 the full name of the GDNF family receptor alpha 1
  • GFRA1 acts as a receptor involved in the transmission of GDNF signaling, mediating signal feedback between the ureteric epithelium and the posterior renal interstitial, and is essential for ureteral buds entering the interstitial tissue. Deletion of the GFRA1 gene results in renal insufficiency or loss in neonatal rats.
  • the use of GFRA1 gene knockout technology can prevent pigs from producing kidneys during development and provide a good development environment for human cell-derived kidneys. Accurate and efficient knockout of the GFRA1 gene in pigs is the first step.
  • 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 GFRA1 gene by CRISPR technology can greatly improve the screening efficiency of genetically engineered pigs with GFRA1 deletion cells and renal developmental defects.
  • the key technical challenge of this path is to design and prepare precisely targeted sgRNAs, because the targeting accuracy of genes is highly dependent on sgRNA target sequences, and the successful design of precisely targeted sgRNAs becomes a key technical issue for knocking out target genes.
  • the present invention is intended to solve this technical problem and thereby provide a solid basis for knocking out the GFRA1 gene.
  • the object of the present invention is to provide a method for CRISPR-Cas9 specific knockdown of the porcine GFRA1 gene and an sgRNA for specifically targeting the GFRA1 gene.
  • the present invention provides an sgRNA for specifically targeting a GFRA1 gene in a CRISPR-Cas9 specific knockout porcine GFRA1 gene, the sgRNA having the following characteristics:
  • the target sequence of the sgRNA on the GFRA1 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 GFRA1 gene is located in the 5 exon coding regions of the N-terminus of the GFRA1 gene, or a part of the target sequence is located at 5 N-terminal exons of the GFRA1 gene, and the rest Partially crossing the boundary with adjacent introns, located adjacent to the intron;
  • the target sequence of the sgRNA on the GFRA1 gene is unique.
  • the above target sequence is the sequence shown by any one of SEQ ID NOS: 1 to 50 in the Sequence Listing.
  • the above target sequence is the sequence shown by SEQ ID NO: 1 or 4 in the Sequence Listing.
  • the present invention provides a method of specifically knocking out a porcine GFRA1 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: 51 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 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 GFRA1 gene is determined by denaturation, renaturation and enzymatic cleavage, specifically:
  • the present invention provides a recombinant expression vector lentiCRISPR v2-GFRA1 for use in a method for CRISPR-Cas9 specific knockout of a porcine GFRA1 gene, the sequence of the backbone vector of the recombinant expression vector being SEQ ID NO: ID NO: 51; 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 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-GFRA1 of the third aspect, in the method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene.
  • the invention specifically targets the CRISPR-Cas9 knock-out porcine GFRA1 gene, and successfully finds the sgRNA which specifically targets the GFRA1 gene, and uses the sgRNA of the present invention in the method of CRISPR-Cas9 specific knockout of the porcine GFRA1 gene, which can be rapidly , accurate, efficient and specific knockout of porcine GFRA1
  • the gene effectively solves the technical problem of constructing a GFRA1 gene knockout pig with a 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 WT indicates that the PCR product of the wild type cell which has not been infected by virus infection and Cas9 is detected by the Cruiser enzyme digestion test.
  • M represents DNA Marker
  • WT indicates that the PCR product of the wild type cell which has not been infected by virus infection and Cas9 is detected by the Cruiser enzyme digestion test.
  • 1 and 4 respectively indicate the targeted cleavage effect of the target sequences No. 1 and No. 4 in Table 1 on the GFRA1 gene, and the arrow indicates a small fragment obtained by cleavage by 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 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.
  • a suitable 20 bp oligonucleotide sequence was searched for as a target sequence in the exon region of the GFRA1 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 double-stranded DNA fragment ie, a double-stranded target sequence oligonucleotide, which may also be referred to as a double-stranded oligonucleotide.
  • 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-GFRA1.
  • 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-GFRA1.
  • a CRISPR pseudotyped lentivirus expressing GFRA1sgRNA was produced using a packaging plasmid, a packaging cell line and a lentiviral CRISPR vector.
  • a pseudotype lentivirus such as lentiCRISPR v2-GFRA1 is 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 GFRA1 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 (porcine) GFRA1 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.
  • 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 coding region of the 5 exons of the N-terminus of the GFRA1 gene, or a part of the target sequence is located at the N-terminus of the GFRA1 gene. The remaining part spans the junction with the adjacent intron and is located in the adjacent intron; the cleavage of such a coding region sequence results in the functional knockout of the GFRA1 gene, and the residual truncated sequence does not form.
  • Functional protein
  • the target sequence is unique on the GFRA1 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:
  • Oligonucleotide sequences can be specifically synthesized by commercial companies (such as Invitrogen) according to the sequences provided. This example and the following examples investigate the knockdown effect of the target sequence shown by the sequences No. 1 and No. 4 listed in Table 1 on the GFRA1 gene.
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the No. 1 target sequence are as follows:
  • AAACGAAGCGGCAGTGCGAAGTACC (SEQ ID NO: 53).
  • the forward oligonucleotide sequence and the reverse oligonucleotide sequence corresponding to the target sequence No. 4 are as follows:
  • AAACGGCGACCCTGTACTTCGCACC (SEQ ID NO: 55).
  • 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: 51 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 to the recovered vector fragment, and The next reaction system is prepared:
  • 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 GFRA1sgRNA
  • 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-GFRA1; 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-GFRA1 false for different target sequences (sequence 1 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.
  • the amplified fragment of interest consists of a sgRNA target sequence with a size of 465 bp.
  • the position of the target sequence to both ends of the fragment is not less than 100 bp.
  • the amplification reaction system (20 ⁇ L) was as follows:
  • the above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.
  • the second to fourth steps are repeated for 35 cycles.
  • the purified DNA fragments are separately denatured and renatured to form hybrid DNA molecules (including mutant samples and wild-type samples).
  • the reaction system is as follows:
  • Genomic PCR fragment 200ng
  • reaction buffer 2 ⁇ L
  • the reaction system has a total of 9 ⁇ L
  • the above reaction system was prepared, placed in a PCR machine, and reacted according to the following procedure.
  • Electrophoresis detection of the digested product detection of target sequence-mediated knockdown of GFRA1 gene.
  • 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. Since the mutant sample contains a part of the wild-type DNA component, it can form a hybrid molecule containing a local ring structure after being renatured.
  • 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 lentiCRISPR v2-GFRA1 pseudotype lentivirus infection target cell based on the target sequence shown in SEQ ID NO:4, four monoclonal cells randomly selected from 100 single cells were detected by Cruiser enzyme electrophoresis, and 4 of them were detected. By cutting small fragments, it is indicated that gene knockout occurs, and the knockout efficiency can reach 100%, indicating that the target sequence shown in sequence 4 has a high target for knocking out the GFRA1 gene.

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Abstract

L'invention concerne un procédé d'utilisation de la spécificité de CRISPR-Cas9 pour inactiver un gène GFRA1 porcin, et un ARNsg utilisé pour cibler de façon spécifique le gène GFRA1. La séquence cible de l'ARNsg permettant le ciblage spécifique du gène GFRA1 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 GFRA1 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 GFRA1.
PCT/CN2015/081232 2015-06-11 2015-06-11 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 WO2016197360A1 (fr)

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PCT/CN2015/081232 WO2016197360A1 (fr) 2015-06-11 2015-06-11 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

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US10077453B2 (en) 2014-07-30 2018-09-18 President And Fellows Of Harvard College CAS9 proteins including ligand-dependent inteins
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US10745677B2 (en) 2016-12-23 2020-08-18 President And Fellows Of Harvard College Editing of CCR5 receptor gene to protect against HIV infection
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US11912985B2 (en) 2020-05-08 2024-02-27 The Broad Institute, Inc. Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence
US12157760B2 (en) 2018-05-23 2024-12-03 The Broad Institute, Inc. Base editors and uses thereof
US12281338B2 (en) 2018-10-29 2025-04-22 The Broad Institute, Inc. Nucleobase editors comprising GeoCas9 and uses thereof

Citations (3)

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

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104498493B (zh) * 2014-12-30 2017-12-26 武汉大学 CRISPR/Cas9特异性敲除乙型肝炎病毒的方法以及用于特异性靶向HBV DNA的gRNA

Patent Citations (3)

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

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK [O] NCBI; 26 May 2015 (2015-05-26), "Trichechus manatus latirostris isolate Lorelei unplaced genomic scaffold, TriManLat1.0 scaffold00031, whole genome shotgun sequence", XP055337411, Database accession no. NW_004443967.1 *

Cited By (48)

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US11795443B2 (en) 2017-10-16 2023-10-24 The Broad Institute, Inc. Uses of adenosine base editors
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