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WO2021125840A1 - Composition pour l'édition de gène ou l'inhibition de son expression, comprenant cpf1 et un guide d'arn-adn chimérique - Google Patents

Composition pour l'édition de gène ou l'inhibition de son expression, comprenant cpf1 et un guide d'arn-adn chimérique Download PDF

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WO2021125840A1
WO2021125840A1 PCT/KR2020/018570 KR2020018570W WO2021125840A1 WO 2021125840 A1 WO2021125840 A1 WO 2021125840A1 KR 2020018570 W KR2020018570 W KR 2020018570W WO 2021125840 A1 WO2021125840 A1 WO 2021125840A1
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dna
target
guide
nucleotide sequence
seq
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이승환
김선욱
김한섭
이위재
박영호
진영배
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Korea Research Institute of Bioscience and Biotechnology KRIBB
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    • C12N15/90—Stable introduction of foreign DNA into chromosome
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/315—Phosphorothioates

Definitions

  • the present invention relates to a composition for genome editing or expression inhibition comprising Cpf1 and a chimeric DNA-RNA guide.
  • CRISPR-Cas12a (hereinafter, Cpf1) is a CRISPR system belonging to class II and type V. Like Cas9 gene scissors, it has a bi-lobed structure of a nuclease domain and a recognition domain, and is a single-stranded crRNA. It is known to bind by forming a DNA-RNA hybrid duplex to the target DNA double helix using Since the discovery of Cas9, Cpf1 has been attracting attention as an excellent gene scissors to complement the limited part of PAM.
  • Cpf1 gene scissors specifically induces DNA double helix cleavage by recognizing a region rich in thymine (T) as PAM (TTTN or TTN) in addition to a region rich in guanine (G) in the intracellular locus. Because of this, it is widely used as target-specific gene scissors. Most recently, there is a trend to enhance the potential as a gene therapy agent by engineering Cpf1 protein to expand the range of targetable genes by extending PAM, or by increasing DNA double helix cleavage efficiency through modification of guide RNA.
  • Cpf1 structurally recognizes a protospacer of 24 bases, and it is known that the internal seed region is approximately 5 to 10 from the PAM sequence.
  • it is more sensitive to mismatch than CRISPR-Cas9, and when the mismatch is introduced into the seed part of the protospacer region, its activity is significantly reduced, so it has been known as a gene scissors with excellent target specificity.
  • it since the possibility of cleavage for mismatches occurring other than the seed part still exists, in the DNA cleavage process including the target nucleotide sequence of Cpf1 with further increased activity later, such off-target cleavage Non-detection issues may be further highlighted.
  • the present inventors completed the present invention by conducting a study to develop the CRISPR-Cpf1 gene scissors having excellent target specificity and improved indel efficiency.
  • Patent Document 1 Korean Patent Publication No. 10-2018-0028996
  • One object of the present invention is a Cpf1 protein or a DNA encoding the same; And to provide a composition for genome editing comprising a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.
  • Another object of the present invention is to provide a method for producing a transformant comprising the step of introducing the composition for genome editing into an isolated cell or organism.
  • Another object of the present invention is to provide a method for altering the expression of a gene product, comprising introducing the composition for genome editing into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product. .
  • Another object of the present invention is an inactive Cpf1 (dCpf1) protein or DNA encoding the same; And to provide a composition for inhibiting gene expression comprising a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.
  • Another object of the present invention is to alter the expression of a gene product, including the step of introducing the composition for genome editing into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product (wherein the DNA molecule is mutant sequence), thereby providing a method for preventing, ameliorating or treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) in a subject.
  • SNP single-nucleotide polymorphism
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same; And to provide a pharmaceutical composition comprising a chimeric DNA-RNA guide or DNA encoding the target nucleotide sequence and a hybridizable nucleotide sequence.
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence, or a pharmaceutical composition using a pharmaceutical composition comprising DNA encoding the same.
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same; and a chimeric DNA-RNA guide including a target nucleotide sequence and a hybridizable nucleotide sequence, or a diagnostic use using a composition for genetic diagnosis including a DNA encoding the same.
  • One aspect of the present invention is a Cpf1 protein or a DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a composition for genome editing comprising DNA encoding the same.
  • Cpf1 is a type V CRISPR system protein, similar to Cas9, a type II CRISPR system protein, in that a single protein binds to crRNA and cuts a target gene, but there is a big difference in how it works.
  • Cpf1 protein works as a single crRNA, there is no need to use crRNA and trans-activating crRNA (tracrRNA) at the same time as in Cas9 or to artificially create a single guide RNA (sgRNA) that combines tracrRNA and crRNA.
  • tracrRNA trans-activating crRNA
  • sgRNA single guide RNA
  • the PAM exists at the 5' position of the target sequence, and the length of the guide RNA that determines the target is also shorter than that of Cas9.
  • Cpf1 has the advantage that genome editing is possible even for a target nucleotide sequence where Cas9 cannot be used, and it is relatively easy to perform compared to Cas9, which produces crRNA, which is a guide RNA.
  • Cpf1 has the advantage that more accurate and various gene editing is possible because a 5' overhang (sticky end) rather than a blunt-end occurs at the position where the target DNA is cut.
  • a technique for more conveniently, accurately and effectively correcting a target genome using the Cpf1 system is provided.
  • the Cpf1 protein is Candidatus genus, Lachnospira genus, Butyrivibrio genus, Peregrinibacteria , Acidominococcus genus, Porphyromonas ( Porphyromonas genus, Prevotella genus, Francisella genus, Candidatus Methanoplasma , or Eubacterium genus, for example, Parcubacteria bacterium (GWC2011_GWC2_44_17) ), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp.
  • BV3L6 Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, Candidatus Paceibacter, Eubacterium eligens, and the like, but are not limited thereto.
  • the Cpf1 protein is Parcubacteria bacterium (GWC2011_GWC2_44_17), Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, or Eubacterium be of eligens derived However, it is not limited thereto.
  • Cpf1 protein derived microorganisms Genbank protein ID NCBI protein GI from NR Database or local GI (for proteins originated from WGS database) Contig ID in WGS database PbCpf1 Parcubacteria bacteriumGWC2011_GWC2_44_17 KKT48220.1 818703647 LCIC01000001.1 PeCpf1 Peregrinibacteriabacterium GW2011_GWA_33_10 KKP36646.1 818249855 LBOR01000010.1 AsCpf1 Acidaminococcus sp.
  • the Cpf1 protein may be isolated from a microorganism or non-naturally occurring by a recombinant method or a synthetic method.
  • the Cpf1 protein may further include a component commonly used for intranuclear delivery of eukaryotic cells (eg, nuclear localization signal (NLS), etc.), but is not limited thereto.
  • the Cpf1 protein may be used in the form of a purified protein, a nucleic acid encoding it (DNA/RNA), or a recombinant vector containing the DNA.
  • the Cpf1 protein is a component commonly used for intranuclear delivery of eukaryotic cells (eg, a nuclear localization signal (NLS; for example, PKKKRKV, KRPAATKKAGQAKKKK, or a nucleic acid molecule encoding the same), etc.).
  • a nuclear localization signal for example, PKKKRKV, KRPAATKKAGQAKKKK, or a nucleic acid molecule encoding the same
  • the Cpf1 protein of the present invention may be used in the form of a purified protein, a nucleic acid encoding the same (DNA/RNA), or a recombinant vector containing the DNA.
  • the Cpf1 protein may be linked with a tag advantageous for isolation and/or purification.
  • a His tag a Flag tag, a small peptide tag such as an S tag, or a Glutathione S-transferase (GST) tag, a Maltose binding protein (MBP) tag, etc. may be used depending on the purpose, but is not limited thereto.
  • GST Glutathione S-transferase
  • MBP Maltose binding protein
  • the guide RNA may be appropriately selected depending on the type of Cpf1 protein to form a complex and/or a microorganism derived therefrom.
  • the term 'genome editing' refers to a nucleic acid molecule (one or more, such as 1-100,000bp, 1-10,000bp, 1 -1000, 1-100bp, 1-70bp, 1-50bp, 1-30bp, 1-20bp, or 1-10bp) loss, alteration, and/or restoration (modification) of gene function by deletion, insertion, substitution, etc. It can be used to mean to let
  • chimeric DNA-RNA guide refers to the substitution of some RNA among the RNAs of crRNA (crispr RNA) specific to the target DNA with DNA, and the chimeric DNA-RNA guide is a complex with Cpf1 protein. can form, and the Cpf1 protein can be brought to the target DNA.
  • DNA substitution of guide RNA can reduce off-target DNA sequence cleavage by changing the binding energy of guide and target DNA.
  • it can be synthesized cheaply, as well as avoiding the issue of 5' phosphate that inevitably occurs during self-production in the laboratory, and avoiding the induction of immune responses. It can be safely used when Cpf1 is introduced.
  • the DNA guide compared to the chemically unstable RNA in aqueous solution, the DNA guide has the advantage of easy chemical modification and easy application access.
  • the DNA may be a substitution at the 3' end of the guide.
  • the DNA is preferably about 6 to 10 substitutions at the 3' end.
  • RNAs from the 3' end in the sequence identified above may be preferably substituted with DNA. More specifically, about 6, 7, 8, 9, or 10 RNAs may be substituted with DNA. Since the chimeric DNA-RNA guide in which 6 to 10 RNAs are substituted with DNA from the 3' end of the crRNA shows high specificity and cleavage activity, it is preferable that the substitution of the crRNA is made at the 3' end of the crRNA.
  • the chimeric DNA-RNA guide in which 6 to 10 RNAs are substituted with DNA from the 3' end of the crRNA shows high specificity and cleavage activity, it is preferable that the substitution of the crRNA is made at the 3' end of the crRNA.
  • hybridization refers to a reaction in which one or more polynucleotides react to form a complex, and the complex is stabilized through hydrogen bonding between bases of nucleotide residues.
  • the nucleotide sequence hybridizable to the gene target site is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% of the nucleotide sequence (target sequence) of the gene target site. It refers to a nucleotide sequence having the above or 100% sequence complementarity (hereinafter, the same meaning is used unless otherwise specified, and the sequence homology can be confirmed using a conventional sequence comparison means (eg, BLAST)) .
  • BLAST sequence comparison means
  • composition for gene editing of the present invention encodes a recombinant vector containing a nucleotide encoding Cpf1 and a recombinant vector containing a nucleotide encoding a chimeric DNA-RNA guide, or a nucleotide encoding Cpf1 and a chimeric DNA-RNA guide It may be introduced into a cell or organism in the form of a recombinant vector containing nucleotides, or it may be introduced into a cell or organism in the form of a mixture containing Cpf1 protein and chimeric DNA-RNA or a ribonucleic acid protein forming a complex thereof.
  • the chimeric DNA-RNA guide of the present invention can hybridize with a target DNA.
  • the composition for gene editing of the present invention can be applied to eukaryotic organisms.
  • the eukaryotic organism is a eukaryotic cell (e.g., a fungus such as yeast, eukaryotic and/or eukaryotic plant-derived cells (e.g., embryonic cells, stem cells, somatic cells, germ cells, etc.), eukaryotic cells (e.g., For example, vertebrates or invertebrates, more specifically humans, primates such as monkeys, mammals including dogs, pigs, cattle, sheep, goats, mice, rats, etc.), and eukaryotic plants (eg, green algae, etc.) may be selected from the group consisting of monocotyledonous or dicotyledonous plants such as algae, corn, soybean, wheat, and rice), but is not limited thereto.
  • a eukaryotic cell e.g., a fungus such as yeast, eukaryotic and/or eukaryotic plant-derived cells
  • composition for gene editing of the present invention encodes a recombinant vector containing a nucleotide encoding Cpf1 and a recombinant vector containing a nucleotide encoding a chimeric DNA-RNA guide, or a nucleotide encoding Cpf1 and a chimeric DNA-RNA guide
  • the recombinant vector may include a promoter operably linked to the nucleotide.
  • promoter is a DNA regulatory region capable of binding to a polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.
  • operably linked refers to a functional linkage (cis) between a gene expression control sequence and another nucleotide sequence.
  • the gene expression control sequence may be at least one selected from the group consisting of a replication origin, a promoter, a transcription terminator, and the like.
  • the promoter of the present invention is one of the transcriptional control sequences that regulate the initiation of transcription of a specific gene, and may be a polynucleotide fragment having a length of typically about 100 bp to about 2500 bp.
  • the promoter can be used without limitation, as long as it can regulate transcription initiation in a cell, for example, a eukaryotic cell (eg, a plant cell, or an animal cell (eg, a mammalian cell such as a human, a mouse, etc.), etc.) Do.
  • a eukaryotic cell eg, a plant cell, or an animal cell (eg, a mammalian cell such as a human, a mouse, etc.), etc.
  • a eukaryotic cell eg, a plant cell, or an animal cell (eg, a mammalian cell such as a human, a mouse, etc.), etc.)
  • the promoter is a CMV promoter (cytomegalovirus promoter; for example, human or mouse CMV immediate-early promoter), U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter , SV40 promoter, adenovirus promoter (major late promoter), pL ⁇ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, vaccinia virus 7.5K promoter, HSV tk promoter, SV40E1 promoter, respiratory syncytial virus (Respiratory) syncytial virus; RSV promoter, metallotionin promoter, ⁇ -actin promoter, ubiquitin C promoter, human interleukin-2 (IL-2) gene promoter, human lymphotoxin gene promoter, human It may be one or more selected from the group consisting of a human granulocyte-macrophage colony stimulating factor (GM)
  • the promoter may be selected from the group consisting of CMV immediate-early promoter, U6 promoter, EF1-alpha (elongation factor 1-a) promoter, EF1-alpha short (EFS) promoter, and the like.
  • the transcription termination sequence may be a polyadenylation sequence (pA) or the like.
  • the origin of replication may be an f1 origin of replication, an SV40 origin of replication, a pMB1 origin of replication, an adeno origin of replication, an AAV origin of replication, or a BBV origin of replication.
  • the vector of the present invention may be selected from the group consisting of viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors.
  • viral vectors such as plasmid vectors, cosmid vectors and bacteriophage vectors, adenoviral vectors, retroviral vectors and adeno-associated viral vectors.
  • Vectors that can be used as the recombinant vector include plasmids used in the art (eg, pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1).
  • phage eg, ⁇ gt4 ⁇ B, ⁇ -Charon, ⁇ z1, M13, etc.
  • viral vectors eg, adeno-associated virus (AAV) vectors, etc. It may be manufactured based on, but is not limited thereto.
  • the production of the recombinant expression vector of the present invention can be prepared using a genetic recombination technique well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art. have.
  • the guide may be a modified 3'-end.
  • the chimeric DNA-RNA guide of the present invention can exhibit high target specificity and excellent indel efficiency without the influence of DNA exonuclease in the cell by modifying the 3'-end.
  • phosphate that is a phosphonate, phosphorothioate or phosphotriester.
  • it may be modified through biotinylation or the like.
  • the chimeric DNA-RNA guide of the present invention can exhibit high target specificity and excellent indel efficiency without the influence of 3' DNA exonuclease in the cell by modifying the 3'-end with phosphorothioate. have.
  • the DNA may be substituted at the 3' end of the guide.
  • the DNA may be 6 to 10.
  • the cleavage activity is low.
  • the chimeric DNA-RNA guide in which 6 to 10 RNAs are substituted with DNA from the 3' end of the crRNA shows high specificity and cleavage activity
  • the substitution of the crRNA is preferably made at the 3' end of the crRNA.
  • the composition may further include SpCas9 nickase (D10A) or inactive (Dead) SpCas9 nickase (D10A or H840A).
  • SpCas9 nickase (D10A) refers to S. pyogenes Cas9 nickase having a D10A mutation.
  • Inactive SpCas9 (dead SpCas9) nickase (D10A or H840A) refers to S. pyogenes Cas9 with D10A, H840A mutations.
  • SpCas9 nickase D10A
  • D10A can remove the negative supercoil of the DNA double helix existing in the cell, it can improve the genome editing efficiency of the chimeric DNA-RNA guide in the cell.
  • Another aspect of the present invention provides a method for producing a transformant comprising the step of introducing the composition for genome editing into an isolated cell or organism.
  • composition for genome editing of the present invention can be introduced into a cell or organism by a method known in the art for introducing a nucleic acid molecule into an organism, cell, tissue or organ, and as is known in the art, suitable according to the host cell This can be done by selecting standard techniques. Such methods include, for example, electroporation, calcium phosphate (CaPO 4 ) precipitation, calcium chloride (CaCl 2 ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic The liposome method and the lithium acetate-DMSO method may be included, but are not limited thereto.
  • Another object of the present invention is to provide a method for altering the expression of a gene product, comprising introducing the composition for genome editing into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product. .
  • Cells that have undergone a nucleic acid alteration event can be isolated using any suitable method.
  • the repair nucleotide molecule further comprises a nucleic acid encoding a selectable marker.
  • selectable markers are well known in the art and nucleic acid sequences encoding these markers are commercially available (see, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989). See).
  • the method using the selection marker that can be visualized by fluorescence can be further sorted using the fluorescence activated cell sorting (FACS) technique.
  • the isolated engineered cells can be used to establish a cell line for transplantation.
  • the isolated altered cells can be cultured using any suitable method to produce a stable cell line.
  • Another object of the present invention is to alter the expression of a gene product, including the step of introducing the composition for genome editing into a cell containing and expressing a DNA molecule having a target sequence and encoding the gene product, whereby mutant or single
  • SNP nucleotide polymorphism
  • a DNA molecule may contain at least one, two, three, four or more SNPs or mutation sites, and the methods described herein may contain at least one, two, three, four or more SNPs or mutation sites of a gene product associated with the alter expression. That is, it is possible to alter the mutant sequence at multiple SNP sites or progenitor SNPs.
  • Diseases for preventing, ameliorating or treating diseases associated with mutations or single-nucleotide polymorphisms (SNPs) in such subjects include, for example, genetic diseases, non-hereditary diseases, viral infections, bacterial infections, cancer, or autoimmune diseases. do.
  • genetic disease refers to a disease caused in part or wholly, directly or indirectly, by one or more abnormalities in the genome, in particular a condition present at birth.
  • the abnormality may be a mutation, insertion or deletion.
  • Inherited diseases include DMD, hemophilia, cystic fibrosis, Huntington's chorea, familial hypercholesterolemia (LDL receptor defect), hepatoblastoma, Wilson's disease, Congenital hepatic porphyria, hereditary disorders of liver metabolism, Lesch Nyhan syndrome, sickle cell anemia, thalassemia, dry skin pigmentation, Fanconi's anemia, retinitis pigmentosa, telangiectasia, Bloom syndrome (Bloom's syndrome), retinoblastoma and Tay-Sachs disease.
  • the non-genetic disease target is to treat the disease by regulating normal genes other than the mutated gene using Cpf1, and may typically be age-related macular degeneration (AMD), but is not limited thereto.
  • AMD age-related macular degeneration
  • the virus, bacterial infection, or a disease caused by them includes, but is not limited to, AIDS, avian flu, influenza, CMV-infected disease, tuberculosis or leprosy.
  • the cancer is bladder cancer, bone cancer, blood cancer, breast cancer, melanoma, thyroid cancer, parathyroid cancer, bone marrow cancer, rectal cancer, throat cancer, laryngeal cancer, lung cancer, esophageal cancer, pancreatic cancer, colorectal cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or It includes any one selected from the group consisting of cervical cancer, gallbladder cancer, oral cancer, colon cancer, perianal cancer, central nervous system tumor, liver cancer, and colorectal cancer, but is not limited thereto.
  • the autoimmune disease is type 1 diabetes, rheumatoid arthritis, celiac disease-sprue, IgA deficiency, Crohn's disease, multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, skin Sclerosis, polymyositis, chronic active hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Grave's disease, myasthenia gravis, autoimmune neutropenia, idiopathic thrombocytopenia Decreased purpura, liver cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's syndrome, pemphigoid bullae, lupus erythematosus, ulcerative colitis or dense deposit disease, and the like.
  • treatment provides a positive therapeutic response to the disease or condition.
  • positive therapeutic response is intended amelioration of a disease or condition, and/or amelioration of symptoms associated with the disease or condition.
  • the improvement of the symptoms includes administration of an effective amount or a therapeutically effective amount of the composition for genome editing.
  • An “effective amount” or “therapeutically effective amount” refers to an amount of an agent sufficient to produce beneficial or desired results.
  • a therapeutically effective amount may vary depending on one or more of the following: the subject and the disease state being treated, the weight and age of the subject, the severity of the disease state, the mode of administration, etc., which can be readily determined by one of ordinary skill in the art. .
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same; And to provide a pharmaceutical composition comprising a chimeric DNA-RNA guide or DNA encoding the target nucleotide sequence and a hybridizable nucleotide sequence.
  • the dosage form of the pharmaceutical composition of the present invention may be for parenteral use.
  • it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.
  • preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • the pharmaceutical composition of the present invention can be administered parenterally, and can be administered via intratumoral administration, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal, intraarterial, intraventricular, intralesional, intrathecal, topical, and combinations thereof. It may be administered by any one route selected from the group consisting of.
  • the dosage of the pharmaceutical composition of the present invention varies depending on the patient's weight, age, sex, health status, diet, administration time, administration method, excretion rate and severity of disease, and may be appropriately selected by those skilled in the art.
  • the pharmaceutical composition of the present invention may be administered at 0.01 ug/kg to 100 mg/kg per day, specifically 1 ug/kg to 1 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. Accordingly, the above dosage does not limit the scope of the present invention in any way.
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence, or a pharmaceutical composition using a pharmaceutical composition comprising DNA encoding the same.
  • the pharmaceutical use may be for preventing, ameliorating or treating a disease associated with a mutation or a single-nucleotide polymorphism (SNP) in a subject. More specifically, for preventing, ameliorating or treating diseases associated with mutations or single-nucleotide polymorphisms (SNPs), including genetic diseases, non-hereditary diseases, viral infections, bacterial infections, cancer, or autoimmune diseases.
  • SNPs single-nucleotide polymorphisms
  • Another object of the present invention is for use in genome editing, Cpf1 protein or DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence capable of hybridizing with a target nucleotide sequence or a composition comprising a DNA encoding the same.
  • Another object of the present invention is for use in the prevention or treatment of diseases associated with mutations or single-nucleotide polymorphisms (SNPs), including genetic diseases, non-genetic diseases, viral infections, bacterial infections, cancer, or autoimmune diseases for, Cpf1 protein or DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence capable of hybridizing with a target nucleotide sequence or a composition comprising a DNA encoding the same.
  • SNPs single-nucleotide polymorphisms
  • Another object of the present invention is to be used to detect cancer, genetic disease, or virus infection by targeting specific DNA or RNA mutations.
  • Cpf1 protein or DNA encoding the same for accurately distinguishing a normal gene from a mutant gene; and a chimeric DNA-RNA guide comprising a nucleotide sequence capable of hybridizing with a target nucleotide sequence or a composition for gene diagnosis comprising a DNA encoding the same.
  • composition for genetic diagnosis according to the present invention can quickly and accurately detect a target gene through excellent target specificity and rapidity.
  • Another object of the present invention is a Cpf1 protein or a DNA encoding the same.
  • a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a composition comprising DNA encoding the same.
  • Another object of the present invention is to prepare a medicament for use in the prevention or treatment of a disease associated with a mutation or single-nucleotide polymorphism (SNP),
  • Cpf1 protein or DNA encoding the same and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable to a target nucleotide sequence or a composition comprising a DNA encoding the same.
  • Another aspect of the present invention is an inactive Cpf1 (dCpf1) protein or a DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a composition for inhibiting gene expression comprising a DNA encoding the same.
  • dCpf1 inactive Cpf1
  • a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a composition for inhibiting gene expression comprising a DNA encoding the same.
  • composition for inhibiting gene expression of the present invention the parts overlapping with the above description may be used in the same meaning as the above description.
  • the deactivated Cpf1 protein can bind to the target gene by the chimeric DNA-RNA guide, but does not show nuclease activity, so the transcription of the target gene may be inhibited or reduced, so it is used to regulate the expression of the target gene. can be used effectively.
  • the inactive Cpf1 protein used in the present invention may be prepared by a method for removing or reducing the activity of the Cpf1 protein known in the art.
  • the guide may have a 3'-end modified with phosphorothioate.
  • the DNA may be substituted at the 3' end of the guide.
  • the composition may further include SpCas9 nickase (D10A).
  • composition for genome editing or expression inhibition comprising Cpf1 and a chimeric DNA-RNA guide
  • the indel efficiency compared to the existing RNA guide is similar, but the target specificity is excellent compared to the RNA guide, so a gene requiring stability and high therapeutic effect It can be effectively applied to treatment.
  • RNA part of crRNA is blue (no underline) and the DNA part is red (underlined). ) (numbers are the number of substituted DNAs).
  • Figure 2 is a chimeric DNA-RNA guide target gene of AsCpf1 (from the 3 'end of the DNA substitutions crRNA unit 4bp) with (DNMT1: Orange (light gray), CCR5 : Green (dark gray)) is a graph showing cutting efficiency.
  • Cr1, Cr2, Cr3, Cr4, Cr5, Cr6, Cr7 and Cr8 represent the chimeric guide sequences of crRNA1, crRNA2, crRNA3, crRNA4, crRNA5, crRNA6, crRNA7 and crRNA8 corresponding to Table 2, respectively, CrS1, CrS2, CrS3, CrS4, CrS5, CrS6, CrS7 and CrS8 represent the chimeric guide sequences of crRNA51, crRNA52, crRNA53, crRNA54, crRNA55, crRNA56, crRNA57 and crRNA58 corresponding to Table 3, respectively.
  • 3 is a graph showing the cleavage efficiency of the target gene ((DNMT1 : target (orange (dark gray))) of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 5' end of crRNA).
  • DNMT1 target (orange (white)), non-target (blue (blue)) for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 3' end of crRNA).
  • DNMT1 target (orange (white)
  • non-target blue (hatched 1:) for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (replacing the seed region of crRNA with DNA).
  • Off-target 1), gray (hatched 2: Off-target 2), yellow (black)) is a graph showing the cutting efficiency.
  • CCR5 target (black), non-target (not confirmed) for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 3' end of crRNA) ) is a graph showing the cutting efficiency.
  • CCR5 target (black), non-target (not confirmed)
  • cleavage efficiency for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide is a graph showing
  • FIG. 9 is a chimeric DNA-RNA guide (3' end 4bp of crRNA, 8bp DNA substitution) using a target gene and a non-target gene ( FANCF : target (black), non-target (not confirmed)) Graph showing the cleavage efficiency to be.
  • FIG. 11 shows a target gene and a non-target gene ( EMX1 : target (dark gray), off-target-1: black (each with a chimeric DNA-RNA guide (3' end 4bp of crRNA, 8bp DNA substitution)) using 16.75, 19.73, 29.50), Off-target 2: Not confirmed) It is a graph showing the cutting efficiency.
  • Figure 12 is (A) a chimeric DNA-RNA guide (from the 3 'end of crRNA DNA substitution of 4bp unit, the number is the substituted DNA repair) of using AsCpf1 DNMT1 target specificity, and (B) a chimeric DNA-RNA guide ( a graph showing the specificity of the target DNMT1 AsCpf1 with the seed region DNA substitution) of crRNA. (meaning DNMT 1 on/off 1 ratio, 2 ratio, 3 ratio from the left in each cell of A and B. For example, based on Cr1(D(0), DNMT 1 on/off 1 from the left) ratio, meaning 2 ratio, 3 ratio)
  • FIG. 13 shows (A) CCR5 target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 3' end of crRNA) and (B) chimeric DNA-RNA guide (DNA substitution in the seed region of crRNA) It is a graph showing the CCR5 target specificity of AsCpf1 using (meaning CCR5 on/off 1 ratio, 2 ratio from the left in each cell of A and B)
  • FIG. 14 is a graph showing (A) FANCF, (B) GRIN2B and (C) EMX1 target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 3' end of crRNA). (On/ Off_1: black, On/Off_2: dark gray)
  • 15 is a diagram illustrating a gene editing pattern using a chimeric DNA-RNA guide.
  • Figure 16 shows a chimeric DNA-RNA guide (3 crRNA 'from the terminal based on the DNA substitution of 4bp unit, the 3' terminal phosphorothioate (phosphorothioate, PS) improvement) DNMT1 gene and the non-target cleavage efficiency with It is a graph (in each cell, from top to bottom, DNMT1 on-target, off-target 1, 2, 3 are in order).
  • 17 is a graph showing the target specificity of a chimeric DNA-RNA guide whose 3' end is modified with PS. (In each cell, from left to right, DNMT1 on/off ratio 1, 2, 3 is in order)
  • 18 is a diagram showing the results of intracellular DNMT1 gene correction using the 3'-end PS-modified chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 5' end of crRNA).
  • 19 is a diagram showing the efficiency of intracellular DNMT1 gene editing using the 3'-end PS-modified chimeric DNA-RNA guide (DNA substitution of 4 bp units from the 5' end of crRNA).
  • 4T represents the extension of 4 thymine bases so as not to inhibit the activity compared to the direct modification of PS at the 3' end of the chimeric guide.
  • 4T-PS indicates that the chimeric guide was modified with PS by extending 4 thymidine bases at the 3' end.
  • Figure 20 is a graph comparing the DNA amplicons with a chimeric DNA-RNA-based guide AsCpf1, LbCpf1 and FnCpf1 gene within a target nucleotide sequence DNMT1 gene of scissors cutting Efficiency.
  • Cr1, Cr2, Cr3, Cr D+9, Cr D+10, Cr D+11, Cr4, Cr5, Cr6, Cr7 and Cr8 are CrRNA1, CrRNA2, CrRNA3, CrRNA-2, CrRNA3-3, CrRNA3 of Table 2, respectively.
  • -4, CrRNA4, CrRNA5, CrRNA6, CrRNA7 and CrRNA8 are shown. (In each cell, from top to bottom, AsCpf1, LbCpf1, FnCpf1 in that order)
  • 21 is a graph showing the cutting efficiency of DNMT1 target gene and a non-target gene of chimeric DNA-RNA guide based FnCpf1 gene scissors. (In each cell, FnCpf1-DNMT1 on, FnCpf1-DNMT1 off1, FnCpf1-DNMT1 off2 from top to bottom)
  • FIG. 22 is a diagram showing a nick created by SpCas9 nickase and a double helix cleavage position induced by CRISPR-Cpf1.
  • 24 shows the results of a target specificity investigation for a gene sequence on a plasmid using a chimeric DNA-RNA guide.
  • 24A shows the comparison results of target/non-target sequence correction efficiency on the plasmid during intracellular (HEK293FT) chimeric DNA-RNA-based AsCpf1 delivery
  • FIG. 24B shows the target for the DNMT1 sequence on the plasmid.
  • Figure 24 C shows the target specificity comparison result for the DNMT1 sequence by next-generation sequencing
  • Figure 24 D shows the target/ratio for the GRIN2B sequence on the plasmid
  • FIG. 24E shows the results of comparison of target specificity with respect to the GRIN2B nucleotide sequence by next-generation sequencing.
  • 25 shows the results of investigation of the Cas12a (Cpf1) gene editing activity by using the CCR5 gene target 3' end PS (phosphorothioate) improved chimeric guide and nickase SpCas9 in combination.
  • 25A shows genome correction by simultaneous delivery of chimeric DNA-RNA-based Cas12a and dead/nickase SpCas9 in HEK293FT cells (Table: CCR5 target gene sequence and in-silico predicted non-target sequence information)
  • B of 25 shows the results of analysis of NGS genome editing efficiency by simultaneous delivery of chimeric DNA-RNA-based Cas12a and nickase SpCas9 (+4DNA: crRNA 3' end 4nt DNA substitution, +8DNA: crRNA 3' end 8nt DNA substitution, PS : phosphorothioate respectively)
  • FIG. 25C shows an increase in genome editing specificity by dead/nickase SpCas9 complex treatment.
  • 26 is a purification result of the gene scissors CRISPR-Cas12a (Cpf1) recombinant protein using an affinity column (Ni-NTA resin). AsCpf1 (Acidaminococcus sp. Cpf1) and LbCpf1 (Cpf1) bound to N-terminus (6X)His-tag were isolated/purified in bacterial cells, respectively, and the result of securing an activated gene scissors protein with a purity of 90% or more indicates.
  • 27A is a Cpf1 target nucleotide sequence for target-specific removal of the CCR5 gene. Red (dark gray) indicates PAM (TTTN) nucleotide sequence, yellow (white) indicates target nucleotide sequence, and B in FIG. 27 is CCR5 target nucleotide sequence (on-target) and similar non-target nucleotide sequence (off-target) ) information (the portion that mismatches the guide RNA in the off-target sequence is indicated by an underscore (G)).
  • NC negative control, control group not treated with gene scissors
  • Only Cas12a Cas12a protein only Treated control group
  • WT crRNA treated with Cas12a using the guide in the form of RNA that was used previously
  • +8DNA crRNA treated with Cas12a in which 8 nt was substituted with DNA at the 3' end of the guide in the form of RNA
  • Cpf1 gene scissors with enhanced target specificity that can cut only the oncogene BRAF (1799T>A) without cutting the normal gene BRAF are applied to remove the oncogene that is the source of cancer cells. It shows a schematic diagram of killing
  • 31 shows a Cpf1 target nucleotide sequence for target-specific removal of an oncogenic BRAF mutant gene.
  • 31A is orange (BRAF_target1): target 24nt nucleotide sequence of CRISPR-Cpf1 gene scissors, blue (TTTN_PAM): CRISPR-Cpf1 gene scissors PAM (TTTN) sequence required for target recognition, red color (1799T->A ): indicates that mutation occurs in the oncogenic gene (1799T>A) in the normal gene (1799T),
  • B of 31 is a point mutation (underline (T)) in the BRAF gene (MT: oncogene, WT : Normal gene (proto-oncogene)).
  • Figure 32 shows the specificity test of the oncogenic BRAF mutation gene compared to the normal gene of Cpf1 using the chimeric DNA-RNA guide (NC: negative control, control group not treated with gene scissors, WT: guide in the form of RNA used previously Cas12a treatment using , D+8: Treatment of Cas12a in which 8 nt is substituted with DNA at the 3' end of the guide in the form of an existing RNA
  • NC negative control, control group not treated with gene scissors
  • WT guide in the form of RNA used previously Cas12a treatment using
  • D+8 Treatment of Cas12a in which 8 nt is substituted with DNA at the 3' end of the guide in the form of an existing RNA
  • Asterisk Cut DNA mark
  • BRAF MT Oncogene BRAF (1799T>A )
  • BRAF WT normal gene BRAF (1799T)
  • 33 shows enhancement of oncogene target specificity of Cpf1 compared to normal genes using a chimeric DNA-RNA guide.
  • 33A shows a comparison experiment of cleavage of the conventional wt-crRNA and 8 DNA substituted chimeric DNA-RNA for the normal gene BRAF (1799T) and the oncogenic gene BRAF (1799T>A), FIG.
  • 33B is A Values calculated from are expressed as target specificity (cleavage efficiency for BRAF(1799T>A)/cleavage efficiency for BRAF(1799T)) (BRAF MT: oncogene BRAF(1799T>A), BRAF WT: normal gene BRAF( 1799T) NC: negative control, control group not treated with gene scissors, WT-crRNA: treated with Cas12a using the previously used RNA guide, D+8-crRNA: 3' end of the existing RNA guide treated with Cas12a with DNA substitution of 8 nt BRAF MT/WT ratio: cleavage target specificity for the oncogene BRAF (1799T>A) compared to the normal gene BRAF (1799T).
  • FIG. 34 shows a Cpf1 target nucleotide sequence for target-specific removal of the VEGFA gene.
  • FIG. 34A shows the target nucleotide sequence in angiogenic endothelial factor (VEGFA) (blue (Cpf1_Target1): target 24nt nucleotide sequence of CRISPR-Cpf1 gene scissors, red (TTTN_PAM): CRISPR-Cpf1 gene scissors PAM (TTTN) sequence required for target recognition).
  • VEGFA angiogenic endothelial factor
  • VEGFA_OT_Site VEGFA target nucleotide sequence
  • nucleotide sequences indicated by underlined in the nucleotide sequence represent mismatched bases different from the Cpf1 guide nucleotide sequence.
  • VEGFA on-target In angiogenic endothelial factor (VEGFA) target nucleotide sequence
  • VEGFA off-target a non-target nucleotide sequence similar to the VEGFA target nucleotide sequence).
  • FIG. 36 shows enhancement of VEGFA gene target specificity compared to non-target Cpf1 using a chimeric DNA-RNA guide.
  • Figure 36 A shows a comparison experiment for cleavage of the existing WT-crRNA and 8 DNA substituted chimeric DNA-RNA for VEGFA on-target and VEGFA off-target
  • B of Figure 36 is the value calculated from A the target It is expressed as specificity (cleavage efficiency for VEGFA on-target / cleavage efficiency for VEGFA off-target)
  • NC negative control, control group not treated with gene scissors
  • WT-crRNA guide in the form of RNA used previously Processed Cas12a used
  • D+8-crRNA Processed Cas12a in which 8 nt was substituted with DNA at the 3' end of the existing RNA guide
  • VEGFA on-target Target nucleotide sequence in angiogenic endothelial factor (VEGFA)
  • VEGFA off-target a non-target sequence similar to the
  • buffer B 20mM Tris-HCl (pH 8.0), 300 nM NaCl
  • bufferC 20mM Tris- HCl (pH 8.0), 300 nM NaCl, 200 mM Imidazole] was used to elute the AsCpf1
  • the eluted protein was exchanged with bufferE [200mM NaCl, 50mM HEPES (pH7.5), 1mM DTT, 40% glycerol] using a centricon (Amicon Ultra,) and aliquoted at -80°C and stored.
  • bufferE 200mM NaCl, 50mM HEPES (pH7.5), 1mM DTT, 40% glycerol
  • the chimeric DNA-RNA guide was synthesized and custom-made (bioneer) according to the target nucleotide sequence (Tables 2 to 4) in each target gene.
  • the 3' ends of the hDNMT1 crRNA2-2, hDNMT1 crRNA2-4, hDNMT1 crRNA3-2 and hDNMT1 crRNA3-4 chimeric guides in Table 4 were modified with phosphorothioate (PS). Bases indicated in bold in Tables 2 to 4 correspond to DNA base sequences.
  • 'Cr + nunber' the chimeric guides of Tables 2 to 4 are briefly indicated as 'Cr + nunber'.
  • 'hDNMT1 crRNA1' was denoted as 'Cr1'
  • 'hFANCF crRNA82-2' was denoted as 'Cr82-2'.
  • the part recognized as a proto-spacer formed by DNA-RNA conjugation was replaced with a DNA sequence in order to reduce the binding energy between the target DNA sequence and the guide RNA ( 1).
  • the guide can be recognized as the overall DNA structure, including the proto-spacer region.
  • RNA-DNA guide Premix the purified recombinant AsCpf1 and the chimeric RNA-DNA guide (Bioneer) or purified crRNA corresponding to each locus synthesized in Example 1, and in the condition of cleavage buffer (NEB3, 10 ⁇ l volumn), 1 Incubated at 37°C for hours. After the reaction, a stop buffer (100 mM EDTA, 1.2% SDS) was added to stop the reaction, and DNA cleavage was confirmed by 2% agarose gel electrophoresis.
  • cleavage buffer 100 mM EDTA, 1.2% SDS
  • the DNA cleavage efficiency was calculated using the imageJ program for the cleaved image pattern and the value of the band intensity profile measured according to the following equation.
  • Example 4 Analysis of cleavage specificity by target gene using a chimeric DNA-RNA guide
  • Example 2 the on-target and off-target cleavage efficiency of Example 2 by selecting a similar nucleotide sequence (off-target) for each gene target nucleotide sequence (on-target) in silico (Cas-offinder) method Each was calculated by the method ( FIGS. 5 to 11 ), and a ratio thereof was calculated ( FIGS. 12 to 14 ).
  • the 3' end of the chimeric DNA-RNA guide is modified with phosphorothioate (PS) to improve the intracellular genome It was confirmed whether the calibration efficiency could be improved.
  • PS phosphorothioate
  • nucleotide sequence cleavage in the DNMT1 gene was induced in vitro using a chimeric DNA-RNA guide (Table 4) modified with PS at the 3' end.
  • a chimeric DNA-RNA guide (Table 4) modified with PS at the 3' end, indels were induced in the same gene sequence.
  • the HEK293FT cell line ATCC was subcultured in DMEM medium (DMEM (Gibco) in 10% FBS (Gibco)) every 48 hours at 37° C. under 5% CO 2 conditions while subculturing 70% confluency. was maintained, and an electroporation kit (amaxa, V4XC-2032) was used for chimeric cell transfection.
  • an electric shock (program: CM-130) was applied in electroporation buffer (Cpf1: 60 pmol, crRNA: 240 pmol) conditions. Thereafter, the transfected cells were transferred to 500 ⁇ l of a DMEM medium solution of a 24-well plate pre-incubated for 30 minutes under 5% CO 2 conditions at 37° C., and cultured under the same conditions (37° C. and 5% CO 2 ).
  • the chimeric RNA-DNA guide and the recombinant AsCpf1 complex were delivered into the cells (HEK293FT) and 48 hours later, genomic DNA was isolated using a genomic DNA purification kit (Qiagen, DNeasy Blood & Tissue Kit).
  • PCR amplicons (DNMT1, CCR5, FANCF, GRIN2B, EMX1 ) are obtained using the DNA primers in Table 5 corresponding to each locus, and denatured with a PCR device. Annealing (gradual decrease in 1 °C increments from 98 °C to 25 °C, 20 minutes) was performed. Purified recombinant T7E1 enzyme (NEB, M0302S) was used in cleavage buffer (50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT), and incubated at 37° C.
  • cleavage buffer 50 mM NaCl, 10 mM Tris-HCl (pH 7.9), 10 mM MgCl 2 , 1 mM DTT
  • nested PCR in order to confirm the correct nucleotide sequence of the editing site of the target gene, nested PCR (denaturation: 98) in PCR amplicons ( DNMT1, CCR5, FANCF, GRIN2B, EMX1 ) obtained using the DNA primers in Table 5 corresponding to the target locus 30 sec at °C, primer annealing: 30 sec at 58 °C, elongation: 30 sec at 72 °C, 35 cycles) was repeated to insert the adapter and index sequences at both ends of the amplicon (denaturation: 30 sec at 98 °C) , primer annealing: 15 s at 62° C., elongation 15 s at 72° C., 35 cycles).
  • the tagged amplicon mixture was loaded into a mini-SEQ analyzer (illumina MiniSeq system, SY-420-1001) according to the manufacturer's instructions, and targeted deep sequencing was performed.
  • the saved Fastq file was analyzed with Cas-Analyzer code and the calibration efficiency (%) was calculated.
  • Example 6 Comparison of DNMT1 target sequence indel induction efficiency in genes of AsCpf1, LbCpf1 and FnCpf1 based on a chimeric DNA-RNA guide using a plasmid
  • LbCpf1 and FnCpf1 were purified to a protein state by expressing a self-made expression plasmid in bacteria in the same manner as in Example 2, as in AsCpf1.
  • a plasmid containing the target DNMT1 sequence was prepared by mimicking the nucleotide sequence on genomic DNA as it is, and it was delivered intracellularly by electroporation like the purified Cpf1 gene scissors protein, and the target DNMT1 sequence contained in the plasmid in the cell was analyzed by the next-generation sequencing method of Example 2.
  • Example 7 Confirmation of improvement of intracellular genome editing efficiency of chimeric DNA-RNA guide-based Cpf1 gene scissors by using SpCas9 nickase (D10A) mixed
  • the negative supercoil was removed and the genome editing efficiency was analyzed.
  • a plasmid expressing SpCas9 nickase (D10A) was delivered to bacteria, purified in protein form, and then mixed with purified guide RNA corresponding to a negative supercoil, a target sequence. This was used together with a chimeric DNA-RNA guide with an unmodified 3' end and a chimeric DNA-RNA guide with a PS-modified 3' end, and the efficiency of intracellular genome editing was measured in the same manner as in Example 5. (Fig. 22).
  • 23A to 23D show the results of confirming the target specificity of the DNMT1, GRIN2B, HPRT1, and RPL32P3 gene sequences, respectively.
  • chimeric DNA-RNA-based AsCpf1 in higher animal cells eg, HEK293FT
  • an experiment was performed to compare the efficiency of target/non-target sequence correction on the plasmid.
  • Example 10 Cas12a (Cpf1) gene editing activity by using the CCR5 gene target 3' end PS (phosphorothioate) improved chimeric guide and nickase SpCas9 combined use
  • Fig. 25B shows the results of analysis of NGS genome editing efficiency by simultaneous delivery of chimeric DNA-RNA-based Cas12a and nickase SpCas9 (+4DNA: crRNA 3' end 4nt DNA substitution, +8DNA: crRNA 3' end 8nt DNA substitution , PS: represents phosphorothioate, respectively).
  • FIG. 25C results of confirming the increase in genome editing efficiency by the dead/nickase SpCas9 complex treatment and the increase in the genome editing specificity by the dead/nickase SpCas9 complex treatment are shown in FIG. 25C .
  • the best effect is obtained when the chimeric guide with the target 3' end PS (phosphorothioate) is improved and the nickase SpCas9 is used in combination with the 8 DNA 3' end sequences according to the present invention. Appearance was confirmed.
  • Example 11 Application of gene therapy for targeted treatment of viral (eg, HIV) infectious disease
  • Ni-NTA resin pre-washed with buffer B [20 mM Tris-HCl (pH 8.0), 300 nM NaCl] and the ultrasonically disrupted intracellular solution were mixed and stirred in a cold room (4C) for 1 hour and 30 minutes.
  • bufferB 20mM Tris-HCl (pH8.0), 300nM NaCl] to remove non-specific binding components through washing with a volume of 10 times
  • bufferC [20mM Tris-HCl (pH8.0), 300 nM NaCl, 200 mM Imidazole] was used to elute the AsCpf1 protein.
  • the eluted protein was exchanged with bufferE [200mM NaCl, 50mM HEPES (pH7.5), 1mM DTT, 40% glycerol] using a centricon (Amicon Ultra,) and aliquoted at -80 ⁇ C and stored. As a result, a protein having high purity DNA cleavage activity was purified, and the results are shown in FIG. 26 .
  • Chimeric DNA-RNA guides required for CCR5 gene targeting experiments were batch-synthesized and custom-made according to the target nucleotide sequence in the target CCR5 gene (bioneer), and the corresponding sequences are shown in Table 6 and FIG. 27 as follows.
  • hCCR5 wt-crRNA
  • GTGGGCAACATGCTGGTCATCCTC TTTT SEQ ID NO: 201
  • hCCR5 chimeric DNA-RNA
  • GTGGGCAACATGCTGGTCATCCTC TTTT SEQ ID NO: 203
  • AAUUUCUACUCUUGUAGAUGUGGGCAACAUGCUGG TCATCCTC 3' SEQ ID NO: 204
  • the Cpf1 target nucleotide sequence for target-specific removal of the CCR5 gene is shown, dark gray indicates PAM (TTTN) nucleotide sequence, and white indicates the target nucleotide sequence.
  • CCR5 target sequence (on-target) and similar off-target sequence information are shown, and parts that mismatch with guide RNA in off-target sequence are underlined.
  • a CCR5 gene targeting experiment of Cpf1 in animal cells using a chimeric DNA-RNA guide was performed.
  • Cpf1 using a specific chimeric DNA-RNA guide with high specificity for cleavage of target DNA we checked whether target-specific genome editing is possible at the cell level.
  • the HEK293FT (ATCC) cell line was cultured.
  • a culture solution containing 10% FBS (Gibco) in DMEM (Gibco) was used, and 70% density of the culture plate was maintained through subculture every 48 hours in an environment of 37 °C and 5% CO2.
  • the experimental results are shown in FIGS. 28 and 29 .
  • the above results show the specificity of the CCR5 gene target in animal cells of the Cpf1 gene scissors by using the chimeric DNA-RNA guide.
  • nickase was used at the same time when using +8DNA crRNA in which the 3' end of guide RNA was replaced with DNA rather than WT crRNA. Efficiency was maximized by treatment (Figs. 28 A and 29 A), whereas the rate of Indel formation by inducing cleavage to a non-target was decreased (Figs. 28 B and 29 B).
  • the target specificity was 2 when using the +8DNA crRNA in which the 3' end of the guide RNA was replaced with DNA rather than the WT crRNA. It was confirmed that it increased more than twice (FIG. 28C).
  • oncogenes Genes with origin that can cause cancer progression in the human body are known as oncogenes.
  • these proto-oncogenes are mutated by reactive oxygen species or radiation, and their properties are converted to oncogenes, they are not controlled by the control system and cause continuous cell division. It proliferates to form cancer. Accordingly, by using the CRISPR-Cpf1 gene scissors with improved target specificity due to reduced non-targeting targeting, the oncogene that causes cancer in humans can be selectively removed compared to the proto-oncogene required in the human body. An experiment was performed to confirm that there is.
  • the Cpf1 gene scissors with improved target specificity that can cut only the oncogene BRAF (1799T>A) without cutting the normal gene BRAF is applied to remove the oncogene that is the source of the cancer cells and kill the cancer cells. Check whether it can be done.
  • the chimeric DNA-RNA guide required for the BRAF mutant gene (1799T>A) target experiment was synthesized and custom-made (bioneer) according to the nucleotide sequence targeting the point mutation (1799T>A) in the target BRAF gene (bioneer). It is shown in Figure 31.
  • hBRAF wt- crRNA
  • hBRAF wt- crRNA
  • hBRAF chimeric DNA-RNA
  • SEQ ID NO: 207 5' AAUUUCUACUCUUGUAGAUGGUCUAGCUACAGAGAAATCTCGA 3'
  • SEQ ID NO: 208 5' AAUUUCUACUCUUGUAGAUGGUCUAGCUACAGAGA AATCTCGA 3'
  • FIG. 31 the Cpf1 target nucleotide sequence for target-specific removal of the oncogenic BRAF mutant gene was specifically described.
  • BRAF_Target1 target 24nt nucleotide sequence of CRISPR-Cpf1 gene scissors
  • TTN_PAM CRISPR-Cpf1 gene scissors
  • TTTN red Color
  • TTN->A red Color (1799T->A
  • MT mutant gene (oncogene)
  • WT normal gene (proto-oncogene) were described.
  • in-vitro cleavage assay was performed. proceeded.
  • the nucleotide sequences of the normal BRAF gene (1799T) and the oncogenic gene BRAF (1799T>A) were synthesized and inserted into the T-vector, and PCR was performed on each containing the target nucleotide sequence to obtain an amplicon.
  • hBRAF_Mutation_amplicon 2 ⁇ g, hBRAF_wild-type_amplicon 2 ⁇ g, AsCpf1 or LbCpf1 protein 2.8 ⁇ g, crRNA (WT) 600ng or chimeric DNA-RNA (D+8) 600ng cleavage buffer (NEB3.1 buffer, deionized water up to 10 ⁇ g) After 1 hour incubation in an incubator at 37°C, the band pattern was confirmed by electrophoresis on agarose 2% gel at 200V-20min. Then, the DNA cleavage efficiency (cleavage efficiency cleaved band intensity/total intensity X100) was calculated with ImageJ software.
  • the DNA cleavage efficiency of Cpf1 gene scissors using wild-type and chimeric DNA-RNA guides (D+8) for the oncogene BRAF (1799T>A) was similar, and for the normal BRAF gene (1799T), the key The DNA cleavage efficiency was significantly reduced when the meric DNA-RNA guide (D+8) was used (FIG. 33 A), so that the Cpf1 gene scissors using the chimeric DNA-RNA guide (D+8) compared to the normal BRAF gene (1799T)
  • the oncogene BRAF (1799T>A) showed increased specificity ( FIG. 33B ).
  • the vascular endothelial factor (VEGFA) gene is directly targeted and fundamentally removed at the DNA level. confirmed that it can be done.
  • Chimeric DNA-RNA guides required for VEGFA gene targeting experiments were batch-synthesized and custom-made according to the target nucleotide sequence in the target VEGFA gene, which is shown in Table 8 and FIG. 34 .
  • hVEGFA wt- crRNA
  • TTTC TGTCCTCAGTGGTCCCAGGCTGCA SEQ ID NO: 209
  • 5'AAUUUCUACUCUUGUAGAUUGUCCUCAGUGGUCCCAGGCUGCA 3' SEQ ID NO: 210
  • hVEGFA chimeric DNA-RNA
  • TTTC TGTCCTCAGTGGTCCCAGGCTGCA SEQ ID NO: 211
  • 5 'AAUUUCUACUCUUGUAGAUUGUCCUCAGUGGUCCC AGGCTGCA 3' SEQ ID NO: 212
  • 34A is a target sequence in angiogenic endothelial factor (VEGFA), respectively, blue (Cpf1_Target1): target 24nt sequence of CRISPR-Cpf1 gene scissors, red (TTTN_PAM): CRISPR-Cpf1 gene scissors are required for target recognition PAM (TTTN) nucleotide sequence is shown.
  • VEGFA angiogenic endothelial factor
  • 34B shows a non-target nucleotide sequence similar to the VEGFA target nucleotide sequence, and nucleotide sequences underlined in the nucleotide sequence represent mismatched bases different from the Cpf1 guide nucleotide sequence, respectively.
  • cleavage efficiency cleaved band intensity/total intensity X100
  • wild-type and chimeric DNA-RNA guide (D) for the nucleotide sequence (on-target) and the non-target nucleotide sequence (off-target) in the target gene as shown in FIG. 35 +8) was compared and analyzed for the cleavage efficiency of Cpf1 using each.

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Abstract

La présente invention concerne une composition pour l'édition d'un gène ou l'inhibition de son expression, comprenant Cpf1 et un guide d'ARN-ADN chimérique. La composition, tout en ayant une efficacité d'indel similaire à celle des guides d'ARN existants, a une excellente spécificité cible par comparaison avec les guides d'ARN et peut ainsi être efficacement appliquée à une thérapie génique, etc. qui nécessite une fiabilité et une efficacité thérapeutique élevée.
PCT/KR2020/018570 2019-12-18 2020-12-17 Composition pour l'édition de gène ou l'inhibition de son expression, comprenant cpf1 et un guide d'arn-adn chimérique Ceased WO2021125840A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024031058A3 (fr) * 2022-08-05 2024-04-25 The Regents Of The University Of California Compositions et procédés pour traiter des maladies neurologiques

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170068400A (ko) * 2015-12-08 2017-06-19 기초과학연구원 Cpf1을 포함하는 유전체 교정용 조성물 및 그 용도
US20180187176A1 (en) * 2016-11-22 2018-07-05 Integrated Dna Technologies, Inc. Crispr/cpf1 systems and methods
KR101896847B1 (ko) * 2017-09-22 2018-09-07 한국생명공학연구원 DNA 절단 활성이 불활성화된 Cpf1을 포함하는 유전자 발현 억제용 조성물 및 그 용도
US20180282722A1 (en) * 2016-11-21 2018-10-04 Massachusetts Institute Of Technology Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11667917B2 (en) * 2017-11-21 2023-06-06 Genkore Co. Ltd. Composition for genome editing using CRISPR/CPF1 system and use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170068400A (ko) * 2015-12-08 2017-06-19 기초과학연구원 Cpf1을 포함하는 유전체 교정용 조성물 및 그 용도
US20180282722A1 (en) * 2016-11-21 2018-10-04 Massachusetts Institute Of Technology Chimeric DNA:RNA Guide for High Accuracy Cas9 Genome Editing
US20180187176A1 (en) * 2016-11-22 2018-07-05 Integrated Dna Technologies, Inc. Crispr/cpf1 systems and methods
KR101896847B1 (ko) * 2017-09-22 2018-09-07 한국생명공학연구원 DNA 절단 활성이 불활성화된 Cpf1을 포함하는 유전자 발현 억제용 조성물 및 그 용도

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HAO YIN, SONG CHUN-QING, SURESH SNEHA, KWAN SUET-YAN, WU QIONGQIONG, WALSH STEPHEN, DING JUNMEI, BOGORAD ROMAN L, ZHU LIHUA JULIE,: "Partial DNA-guided Cas9 enables genome editing with reduced off-target activity", NATURE CHEMICAL BIOLOGY, NATURE PUB. GROUP, NEW YORK, vol. 14, no. 3, New York, pages 311 - 316, XP055515605, ISSN: 1552-4450, DOI: 10.1038/nchembio.2559 *
KIM HANSEOP, LEE WI-JAE, OH YEOUNSUN, KANG SEUNG-HUN, HUR JUNHO K, LEE HYOMIN, SONG WOOJEUNG, LIM KYUNG-SEOB, PARK YOUNG-HO, SONG : "Enhancement of target specificity of CRISPR-Cas12a by using a chimeric DNA-RNA guide", NUCLEIC ACIDS RESEARCH, OXFORD UNIVERSITY PRESS, GB, vol. 48, no. 15, 4 September 2020 (2020-09-04), GB, pages 8601 - 8616, XP055824405, ISSN: 0305-1048, DOI: 10.1093/nar/gkaa605 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024031058A3 (fr) * 2022-08-05 2024-04-25 The Regents Of The University Of California Compositions et procédés pour traiter des maladies neurologiques

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