WO2021125840A1 - Composition for editing gene or inhibiting expression thereof, comprising cpf1 and chimeric dna-rna guide - Google Patents

Abstract

The present invention relates to a composition for editing a gene or inhibiting the expression thereof, comprising Cpf1 and a chimeric DNA-RNA guide. The composition, while having an indel efficiency similar to that of existing RNA guides, has excellent target specificity compared to the RNA guides, and thus can be effectively applied to gene therapy, etc. that requires reliability and high therapeutic efficacy.

Description

Composition for genome editing or expression inhibition comprising CPF1 and chimeric DNA-RNA guide

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. In addition, 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.

To date, 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. In the process of recognizing the target DNA, 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. . However, 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.

Accordingly, 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.

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. In particular, since 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. Also, unlike Cas9, in the Cpf1 system, 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. Utilizing these features, 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. In addition, 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. In the present specification, a technique for more conveniently, accurately and effectively correcting a target genome using the Cpf1 system is provided.

For example, 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. In one embodiment, 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.

Examples of the Cpf1 protein as described above are summarized in Table 1 below for each derived microorganism:

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. BVBLG WP_021736722.1 545612232 AWUR01000016.1 PmCpf1 Porphyromonas macacae WP_018359861.1 517171043 BAKQ01000001.1 LbCpi1 Lachnospiraceae bacterium ND2006 WP_035635841.1 737666241 JNKS01000011.1 pccpf1 Porphyromonas crevioricanis WP_036890108.1 739008549 JQJC01000021.1 PdCpf1 Prevotella disiens WP_004356401.1 490490171 AEDO01000031.1 MbCpf1 Moraxella bovoculi 237 KDN25524.1 639140168 AOMT01000011.1 LiCpf1 Leptospira inadai WP_020988726.1 537834683 AHMM02000017.1 Lb2Cpf1 Lachnospiraceae bacteriumMA2020 WP_044919442.1 769142322 JQKK01000008.1 FnCpf1 Francisella novicida U112 WP_003040289.1 489130501 CP000439.1 CMtCpf1 Candidatus Methanoplasmatermitum AIZ56868.1 851218172 CP010070.1 EeCpf1 Eubacterium eligens WP_012739647.1 502240446 CP001104.1

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.

Specifically, 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.). ) may be further included at the N-terminus or C-terminus (or at the 5' end or 3' end of the nucleic acid molecule encoding the same), but is not limited thereto. 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. For example, 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.

The guide RNA may be appropriately selected depending on the type of Cpf1 protein to form a complex and/or a microorganism derived therefrom.

As used herein, the term 'genome editing', unless otherwise specified, 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

As used herein, the term "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. In addition, 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. In addition, compared to the chemically unstable RNA in aqueous solution, the DNA guide has the advantage of easy chemical modification and easy application access.

In such a chimeric DNA-RNA guide, the DNA may be a substitution at the 3' end of the guide. Specifically, the DNA is preferably about 6 to 10 substitutions at the 3' end.

For example, about 6 to 10 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.

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.

As used herein, the term “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.

In the present specification, 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)) .

The 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.

The 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 When used in the form of a recombinant vector containing a nucleotide, the recombinant vector may include a promoter operably linked to the nucleotide.

As used herein, the term "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.

As used herein, the term "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. For example, 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 λ 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, β-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-CSF) gene promoter, and the like, but is not limited thereto. In one example, 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. 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). , pHV14, pGEX series, pET series, pUC19, etc.), phage (eg, λgt4λB, λ-Charon, λΔz1, M13, etc.) or 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.

According to one embodiment of the present invention, 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.

Specifically, it may include modification with a phosphate that is a phosphonate, phosphorothioate or phosphotriester. Also LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2' - a modification selected from the group consisting of hydroxyl and combinations thereof. In addition, it may be modified through biotinylation or the like.

Specifically, it may be modified with phosphorothioate.

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.

According to one embodiment of the present invention, the DNA may be substituted at the 3' end of the guide.

According to one embodiment of the present invention, the DNA may be 6 to 10.

In the case of a chimeric DNA-RNA guide in which the 5' end of crRNA or RNA in the seed region is substituted with DNA, the cleavage activity is low. On the other hand, 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, the substitution of the crRNA is preferably made at the 3' end of the crRNA.

According to one embodiment of the present invention, 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.

Since SpCas9 nickase (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.

The 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 (ie, “altered” cells) can be isolated using any suitable method. The repair nucleotide molecule further comprises a nucleic acid encoding a selectable marker. Such 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 To provide a method for preventing, ameliorating or treating a disease associated with a nucleotide polymorphism (SNP). wherein the DNA molecule comprises a mutant or SNP mutant sequence.

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.

Herein, "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. Can affect coding sequence of gene or its regulatory sequence.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.

Here, 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.

Here, 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.

Here, 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.

In the context of the above, treatment provides a positive therapeutic response to the disease or condition. By “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. In the case of formulation, it is prepared using diluents or excipients, such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. In particular, 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. can For a desirable effect, 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.

Here, 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. can

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.

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.

The 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; and

To provide a genome editing use of 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.

In the 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.

According to one embodiment of the present invention, the guide may have a 3'-end modified with phosphorothioate.

According to one embodiment of the present invention, the DNA may be substituted at the 3' end of the guide.

According to one embodiment of the present invention, the composition may further include SpCas9 nickase (D10A).

According to a 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.

1 is a schematic diagram showing the interaction between crRNA and target DNA single-stranded in AsCpf1 protein. To check the performance of crRNA partial DNA substitution, the 3' end, 5' end or seed region was substituted with DNA. The 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).

Figure 4 using the various types of crRNA from replacing crRNA for the nearest seed region with DNA on PAM (TTTN) base sequence at the connection portion of the crRNA portion and a target gene DNA in AsCpf1, the target gene (DNMT1: orange (white ), CCR5 : It is a graph showing the cutting efficiency of green (hatched)).

5 is a target gene ( 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). Shaded 1: off-target 1), gray (hatched 2: Off-target 2), yellow (black))) is a graph showing the cutting efficiency.

6 is a target gene ( 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.

7 is a target gene (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.

8 is a target gene (CCR5 : target (black), non-target (not confirmed)) cleavage efficiency for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (replacing the seed region of crRNA with DNA) is a graph showing

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.

10 shows a target gene and a non-target gene ( GRIN2B : target (dark gray), off-target-1: not identified, using a chimeric DNA-RNA guide (4 bp at the 3' end of crRNA, 8 bp DNA substitution). Off-target-2: black (20.89, 24.51, 13.90, respectively))))) is a graph showing the cutting efficiency.

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)

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)

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)

22 is a diagram showing a nick created by SpCas9 nickase and a double helix cleavage position induced by CRISPR-Cpf1.

23 is a chimeric DNA-RNA guide (4nt at the 3' end of crRNA, 8nt length of consecutive DNA substitutions, +4, +8 for DNMT1, GRIN2B, HPRT1, RPL32P3 (A, B, C, D, respectively)) The results of comparison of the target specificity of the target gene sequence of AsCpf1 using the number of substituted DNAs) are shown (On/Off1 ratio (specificity)).

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, and FIG. 24B shows the target for the DNMT1 sequence on the plasmid. / Shows the comparison result of non-target sequence correction efficiency, Figure 24 C shows the target specificity comparison result for the DNMT1 sequence by next-generation sequencing, and Figure 24 D shows the target/ratio for the GRIN2B sequence on the plasmid The results of comparison of target sequencing efficiency are shown, and 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), and 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)).

28 shows the results of enhancing the specificity of the CCR5 gene target compared to non-target in animal cells (HEK293FT) of AsCpf1 using a chimeric DNA-RNA guide (NC: negative control, control group not treated with gene scissors, Only Cas12a: Cas12a protein Control experimental group treated only with WT crRNA: Cas12a using the guide in the form of RNA that was used previously was treated, +8DNA crRNA: In the guide in the form of RNA, Cas12a in which 8 nt was substituted with DNA at the 3' end was treated )

29 shows enhancement of CCR5 gene target specificity compared to non-target in animal cells (HEK293FT) of LbCpf1 using chimeric DNA-RNA guide (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) .

30 is a target-specific oncogene removal principle. 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 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).

34 shows a Cpf1 target nucleotide sequence for target-specific removal of the VEGFA gene. Specifically, 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). 34B is a non-target nucleotide sequence similar to the VEGFA target nucleotide sequence (VEGFA_OT_Site), and the nucleotide sequences indicated by underlined in the nucleotide sequence represent mismatched bases different from the Cpf1 guide nucleotide sequence.

35 shows Cpf1 non-target versus VEGFA gene specificity experiment using a chimeric DNA-RNA guide (NC: negative control, control group not treated with gene scissors, WT: Cas12a using the previously used RNA guide) Treatment, D+8: Treated with Cas12a replacing 8 nt with DNA at the 3' end of the guide in the form of RNA. Asterisk: Display of cut DNA. 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 VEGFA target sequence).

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these examples.

Example 1. Purification of Cpf1 recombinant protein and synthesis of chimeric DNA-RNA guide

In order to synthesize a chimeric DNA-RNA guide, first, among known Cpf1 proteins, AsCpf1 ( Acidaminococcus sp. Cpf1) recombinant protein whose interaction with the guide RNA has been well defined was purified.

Specifically, The pET28a-Cas12a(AsCpf1) bacterial expression vector was transformed into E. coli BL21(DE3) species, and then incubated at 37°C until OD=0.6. After IPTG inoculation, bacterial cells were precipitated 48 hours later to remove the culture medium, and the remaining precipitate was treated with bufferA [20mM Tris-HCl (pH8.0), 300mM NaCl, 10mM β-mercaptoethanol, 1% TritonX-100, 1mM PMSF]. was redissolved. Thereafter, the bacterial cell membrane was broken by sonication on ice for 3 minutes, and a cell lysate was harvested after centrifugation (5000 rpm, 10 min). Then, the 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 1 hour 30 minutes at 4 ° C. stirred for a while. After bacterial cell precipitation, bufferB [20mM Tris-HCl (pH8.0), 300nM NaCl] was used, washed with 10 times volume to remove non-specific binding components, and bufferC [20mM Tris- HCl (pH 8.0), 300 nM NaCl, 200 mM Imidazole] was used to elute the AsCpf1 protein to purify the protein having high purity DNA cleavage activity. 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℃ and stored.

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.

In the drawings, the chimeric guides of Tables 2 to 4 are briefly indicated as 'Cr + nunber'. For example, 'hDNMT1 crRNA1' was denoted as 'Cr1', and 'hFANCF crRNA82-2' was denoted as 'Cr82-2'.

target gene location division Base sequence (5'→3') SEQ ID NO: hDNMT1
crRNA1 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 1 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 2 hDNMT1
crRNA2 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 3 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUA CTCG SEQ ID NO: 4 hDNMT1
crRNA3 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 5 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCU GTTACTCG SEQ ID NO: 6 hDNMT1
crRNA3-2 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 7 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUC UGTTACTCG SEQ ID NO: 8 hDNMT1
crRNA3-3 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 9 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGU CUGTTACTCG SEQ ID NO: 10 hDNMT1
crRNA3-4 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 11 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUG UCUGTTACTCG SEQ ID NO: 12 hDNMT1
crRNA4 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 13 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAU GTCTGTTACTCG SEQ ID NO: 14 hDNMT1
crRNA5 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 15 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGU CCATGTCTGTTACTCG SEQ ID NO: 16 hDNMT1
crRNA6 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 17 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGA TGGTCCATGTCTGTTACTCG SEQ ID NO: 18 hDNMT1
crRNA7 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 19 Chimeric Guide AAUUUCUACUCUUGUAGAU CTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 20 hDNMT1
crRNA8 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 21 Chimeric Guide AATTTCTACTCTTGTAGATCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 22 hDNMT1
crRNA9 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO:23 Chimeric Guide AATT UCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 24 hDNMT1
crRNA10 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 25 Chimeric Guide AATTTCTA CUCUUGUAGAUCUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 26 hDNMT1
crRNA11 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 27 Chimeric Guide AATTTCTACTCT UGUAGAUCUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 28 hDNMT1
crRNA12 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 29 Chimeric Guide AATTTCTACTCTTGTA GAUCUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 30 hDNMT1
crRNA13 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 31 Chimeric Guide AATTTCTACTCTTGTAGAT CUGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 32 hDNMT1
crRNA14 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 33 Chimeric Guide AAUUUCUACUCUUGUAGAU CTGA UGGUCCAUGUCUGUUACUCG SEQ ID NO: 34 hDNMT1
crRNA15 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 35 Chimeric Guide AAUUUCUACUCUUGUAGAU CTGATGGT CCAUGUCUGUUACUCG SEQ ID NO: 36 hDNMT1
crRNA16 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 37 Chimeric Guide AAUUUCUACUCUUGUAGAU CTGATGGTCCAT GUCUGUUACUCG SEQ ID NO: 38 hDNMT1
crRNA17 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 39 Chimeric Guide AAUUUCUACUCUUGUAGAU C UGAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 40 hDNMT1
crRNA18 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 41 Chimeric Guide AAUUUCUACUCUUGUAGAUC T GAUGGUCCAUGUCUGUUACUCG SEQ ID NO: 42 hDNMT1
crRNA19 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 43 Chimeric Guide AAUUUCUACUCUUGUAGAUCU G AUGGUCCAUGUCUGUUACUCG SEQ ID NO: 44 hDNMT1
crRNA20 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 45 Chimeric Guide AAUUUCUACUCUUGUAGAUCUG A UGGUCCAUGUCUGUUACUCG SEQ ID NO: 46 hDNMT1
crRNA21 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 47 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGA T GGUCCAUGUCUGUUACUCG SEQ ID NO: 48 hDNMT1
crRNA22 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 49 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAU G GUCCAUGUCUGUUACUCG SEQ ID NO: 50 hDNMT1
crRNA23 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 51 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUG G UCCAUGUCUGUUACUCG SEQ ID NO: 52 hDNMT1
crRNA24 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 53 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGG T CCAUGUCUGUUACUCG SEQ ID NO: 54 hDNMT1
crRNA25 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 55 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGU C CAUGUCUGUUACUCG SEQ ID NO: 56 hDNMT1
crRNA26 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 57 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUC C AUGUCUGUUACUCG SEQ ID NO: 58 hDNMT1
crRNA27 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 59 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCC A UGUCUGUUACUCG SEQ ID NO: 60

target gene location division Base sequence (5'→3') SEQ ID NO: hCCR5
crRNA51 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 61 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGGGUGGAACAAGAUGGAU SEQ ID NO: 62 hCCR5
crRNA52 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 63 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGGGUGGAACAAGAU GGAT SEQ ID NO: 64 hCCR5
crRNA53 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 65 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGGGUGGAACA AGATGGAT SEQ ID NO: 66 hCCR5
crRNA54 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 67 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGGGUGG AACAAGATGGAT SEQ ID NO: 68 hCCR5
crRNA55 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 69 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGG GTGGAACAAGATGGAT SEQ ID NO: 70 hCCR5
crRNA56 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 71 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCA CAGGGTGGAACAAGATGGAT SEQ ID NO: 72 hCCR5
crRNA57 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 73 Chimeric Guide AAUUUCUACUCUUGUAGAU TGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 74 hCCR5
crRNA58 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 75 Chimeric Guide AATTTCTACTCTTGTAGATTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 76 hCCR5
crRNA61 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 77 Chimeric Guide AAUUUCUACUCUUGUAGAU T GCACAGGGUGGAACAAGAUGGAU SEQ ID NO: 78 hCCR5
crRNA62 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 79 Chimeric Guide AAUUUCUACUCUUGUAGAUU G CACAGGGUGGAACAAGAUGGAU SEQ ID NO: 80 hCCR5
crRNA63 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 81 Chimeric Guide AAUUUCUACUCUUGUAGAUUG C ACAGGGUGGAACAAGAUGGAU SEQ ID NO: 82 hCCR5
crRNA64 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 83 Chimeric Guide AAUUUCUACUCUUGUAGAUUGC A CAGGGUGGAACAAGAUGGAU SEQ ID NO: 84 hCCR5
crRNA65 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 85 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCA C AGGGUGGAACAAGAUGGAU SEQ ID NO: 86 hCCR5
crRNA66 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 87 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCAC A GGGUGGAACAAGAUGGAU SEQ ID NO: 88 hCCR5
crRNA67 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 89 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACA G GGUGGAACAAGAUGGAU SEQ ID NO: 90 hCCR5
crRNA68 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 91 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAG G GUGGAACAAGAUGGAU SEQ ID NO: 92 hCCR5
crRNA69 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO:93 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGG G UGGAACAAGAUGGAU SEQ ID NO: 94 hCCR5
crRNA70 AsCpf1 target TTTGTGCACAGGGTGGAACAAGATGGAT SEQ ID NO: 95 Chimeric Guide AAUUUCUACUCUUGUAGAUUGCACAGGG T GGAACAAGAUGGAU SEQ ID NO: 96 hFANCF
crRNA81 AsCpf1 target TTTGGTCGGCATGGCCCCATTCGCACGG SEQ ID NO: 97 Chimeric Guide AAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAUUCGCACGG SEQ ID NO: 98 hFANCF
crRNA82 AsCpf1 target TTTGGTCGGCATGGCCCCATTCGCACGG SEQ ID NO: 99 Chimeric Guide AAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAUUCGC ACGG SEQ ID NO: 100 hFANCF
crRNA82-2 AsCpf1 target TTTGGTCGGCATGGCCCCATTCGCACGG SEQ ID NO: 101 Chimeric Guide AAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAU TCGCACGG SEQ ID NO: 102 hFANCF
crRNA83 AsCpf1 target TTTGGTGCTCAATGAAAGGAGATAAGGT SEQ ID NO: 103 Chimeric Guide AAUUUCUACUCUUGUAGAUGUGCUCAAUGAAAGGAGAUAAGGU SEQ ID NO: 104 hFANCF
crRNA84 AsCpf1 target TTTGGTGCTCAATGAAAGGAGATAAGGT SEQ ID NO: 105 Chimeric Guide AAUUUCUACUCUUGUAGAUGUGCUCAAUGAAAGGAGAUA AGGT SEQ ID NO: 106 hFANCF
crRNA84-2 AsCpf1 target TTTGGTGCTCAATGAAAGGAGATAAGGT SEQ ID NO: 107 Chimeric Guide AAUUUCUACUCUUGUAGAUGUGCUCAAUGAAAGGA GATAAGGT SEQ ID NO: 108 hEMX1
crRNA85 AsCpf1 target TTTGTCCTCCGGTTCTGGAACCACACCT SEQ ID NO: 109 Chimeric Guide AAUUUCUACUCUUGUAGAUUCCUCCGGUUCUGGAACCACACCU SEQ ID NO: 110 hEMX1
crRNA86 AsCpf1 target TTTGTCCTCCGGTTCTGGAACCACACCT SEQ ID NO: 111 Chimeric Guide AAUUUCUACUCUUGUAGAUUCCUCCGGUUCUGGAACCAC ACCT SEQ ID NO: 112 hEMX1
crRNA86-2 AsCpf1 target TTTGTCCTCCGGTTCTGGAACCACACCT SEQ ID NO: 113 Chimeric Guide AAUUUCUACUCUUGUAGAUUCCUCCGGUUCUGGA ACCACACCT SEQ ID NO: 114

target gene location division Base sequence (5'→3') SEQ ID NO: hDNMT1
crRNA2-2 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 115 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUA CTCG SEQ ID NO: 116 hDNMT1
crRNA2-3 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 117 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUA CTCGTTTT SEQ ID NO: 118 hDNMT1
crRNA2-4 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 119 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCUGUUA CTCGTTT SEQ ID NO: 120 hDNMT1
crRNA3-2 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 121 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCU GTTACTCG SEQ ID NO: 122 hDNMT1
crRNA3-3 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 123 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCU GTTACTCGTTTT SEQ ID NO: 124 hDNMT1
crRNA3-4 AsCpf1 target TTTCCTGATGGTCCATGTCTGTTACTCG SEQ ID NO: 125 Chimeric Guide AAUUUCUACUCUUGUAGAUCUGAUGGUCCAUGUCU GTTACTCGTTT SEQ ID NO: 126

Example 2. Analysis of genome cleavage efficiency according to partial DNA substitution of Cpf1 guide RNA

Based on the structural model suggesting the interaction of Cpf1 protein and guide, 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). In addition, it was also confirmed whether the guide can be recognized as the overall DNA structure, including the proto-spacer region. When four guide sequences were sequentially replaced with DNA starting from the 3' end, cleavage efficiency was compared and analyzed for target gene DNA (DNMT1, CCR5).

Specifically, each of the above to obtain a gene (DNMT1, CCR5) DNA primers (Table 5) PCR Amplifier silicon (amplicon) (DNMT1, CCR5, FANCF, GRIN2B, EMX1) from the genomic DNA (HEK293FT) using for the. The nucleotide sequences of the forward adapter and reverse adapter primers used for next-generation sequencing (NGS) are shown in bold, respectively.

primer Primer sequence (5'→3') SEQ ID NO: hDNMT1 on-target F1 GTTGCACGTGTCAAGTGCTTA SEQ ID NO: 127 hDNMT1 on-target R1 TTAAAATCCAGAATGCACAAAGTACT SEQ ID NO: 128 hDNMT1 on-target F2 GTGAATTTGGCTCAGCAGGCA SEQ ID NO: 129 hDNMT1 on-target R2 AAGCGAACCTCACACAACAGC SEQ ID NO: 130 hDNMT1 off-target1 F1 TTGACGGCAGTATTACAGGTAG SEQ ID NO: 131 hDNMT1 off-target1 R1 AAGGTCAAATGCCGTTTAACCA SEQ ID NO: 132 hDNMT1 off-target1 F2 GTAGTCAGGCATGAGTGGCA SEQ ID NO: 133 hDNMT1 off-target1 R2 TGCCTCTTTCCCAGGATTCT SEQ ID NO: 134 hDNMT1 off-target2 F1 TCTCAGGCAAGTCACAACTCT SEQ ID NO: 135 hDNMT1 off-target2 R1 TAGGCACATGAAGGTCAAATGC SEQ ID NO: 136 hDNMT1 off-target2 F2 GTTACAGGTAGTTAAGCAGGCA SEQ ID NO: 137 hDNMT1 off-target2 R2 AAATGCCATATTTAACCGTGATCCT SEQ ID NO: 138 hDNMT1 off-target3 F1 TCATGCCTTTCTGGGTCTCAT SEQ ID NO: 139 hDNMT1 off-target3 R1 CACCTGACCTCTGTCACTTTA SEQ ID NO: 140 hDNMT1 off-target3 F2 CTGTTCAGGGAATGGAAAGTGA SEQ ID NO: 141 hDNMT1 off-target3 R2 CTACATCTACGCTCTCCCCA SEQ ID NO: 142 hCCR5 on-target F1 AAGGCTGAGCTGCACCATGC SEQ ID NO: 143 hCCR5 on-target R1 AGGATGATGAAGAAGATTCCA SEQ ID NO: 144 hCCR5 on-target F2 CATTCACTCCATGGTGCTATA SEQ ID NO: 145 hCCR5 on-target R2 ATAGAGCCCTGTCAAGAGTTGA SEQ ID NO: 146 hCCR5 off-target1 F1 AGAAATAGGAGTCTTCATGCCC SEQ ID NO: 147 hCCR5 off-target1 R1 TGGGGTCAATGAGAGGAGTATT SEQ ID NO: 148 hCCR5 off-target1 F2 TAGCAAGGAACCTCAAAGTGC SEQ ID NO: 149 hCCR5 off-target1 R2 CTGCTTCCAATACAATCCACAC SEQ ID NO: 150 hCCR5 off-target2 F1 TAGTGCTGAAAGCTCAGAGAG SEQ ID NO: 151 hCCR5 off-target2 R1 TTCGAGAGCCACACATGAAG SEQ ID NO: 152 hCCR5 off-target2 F2 AGAGAGGGGGTCATAGACTTT SEQ ID NO: 153 hCCR5 off-target2 R2 ACCTTCCAGCCGGTATATTAAT SEQ ID NO: 154 hFANCF on-target F1 TGAAAGCGGAAGTAGGGCCT SEQ ID NO: 155 hFANCF on-target R1 CTCCGCCTGGGTCTTCATCA SEQ ID NO: 156 hFANCF on-target F2 GGAAGTAGGGCCTTCGCGCA SEQ ID NO: 157 hFANCF on-target R2 CCTCCTGGAGATTTGGGTTC SEQ ID NO: 158 hFANCF off-target1 F1 CCTGTAAGCCCTAAGCAAGTC SEQ ID NO: 159 hFANCF off-target1 R1 GTCAAGTTCTACTCAAGGCCAT SEQ ID NO: 160 hFANCF off-target1 F2 CTGTCCTGTAGAGACTTCTGG SEQ ID NO: 161 hFANCF off-target1 R2 GTGGTGGAGGGAAGCTTATTTT SEQ ID NO: 162 hFANCF off-target2 F1 TGGGGTAGGTCTTCAGGAAA SEQ ID NO: 163 hFANCF off-target2 R1 CGTTCTAAATTCTCTACAGTCAAC SEQ ID NO: 164 hFANCF off-target2 F2 TCGTGCTAAGTTACCGAGTT SEQ ID NO: 165 hFANCF off-target2 R2 GCAATAGTTTTGAGCCAGTGA SEQ ID NO: 166 hGRIN2B on-target F1 ACCTCTGCTGAGCACGTTTT SEQ ID NO: 167 hGRIN2B on-target R1 GACAGCAATGCCAATGCTGG SEQ ID NO: 168 hGRIN2B on-target F2 CTCACTTTGTCTGGCCTTGC SEQ ID NO: 169 hGRIN2B on-target R2 CTTCTGAGAACGAGCTCTGC SEQ ID NO: 170 hGRIN2B off-target1 F1 TGACTGCAATTTTGGGTTCCATC SEQ ID NO: 171 hGRIN2B off-target1 R1 GCCACTGCCATTTATTATGTAAC SEQ ID NO: 172 hGRIN2B off-target1 F2 GTCTAATTACACTTGCCATACCT SEQ ID NO: 173 hGRIN2B off-target1 R2 GAAATCCAGCTGTGACTAAATATG SEQ ID NO: 174 hGRIN2B off-target2 F1 GGAGCACAATGAGTGTTTTT SEQ ID NO: 175 hGRIN2B off-target2 R1 GTCTTTTCCTGGTCACATGG SEQ ID NO: 176 hGRIN2B off-target2 F2 GCCAGAAATGTTATTCCATAGTAG SEQ ID NO: 177 hGRIN2B off-target2 R2 CAGTGTTCTTACATATCAGACAC SEQ ID NO: 178 hEMX1 on-target F1 ACTACTCACATCCACTCTGTGAAG SEQ ID NO: 179 hEMX1 on-target R1 GAGTGGCCAGAGTCCAGCTT SEQ ID NO: 180 hEMX1 on-target F2 TAGAGGAGCTAGGATGCACAGCA SEQ ID NO: 181 hEMX1 on-target R2 GCAGCAAGCAGCACTCTGCC SEQ ID NO: 182 hEMX1 off-target1 F1 CAGAGCCTGGAGAATTGATAG SEQ ID NO: 183 hEMX1 off-target1 R1 CTGGGGAGCAGAATAAATTATTG SEQ ID NO: 184 hEMX1 off-target1 F2 GAATAGACCTGGGATGTGCAG SEQ ID NO: 185 hEMX1 off-target1 R2 GGAACCTGAAGAATGGGATTG SEQ ID NO: 186 hEMX1 off-target2 F1 AGATTGAGATCATCCACCCT SEQ ID NO:187 hEMX1 off-target2 R1 AAAACAGAGAGGTTGAGTCTC SEQ ID NO: 188 hEMX1 off-target2 F2 TGTCCACCATGTCCCAGAAG SEQ ID NO: 189 hEMX1 off-target2 R2 ACAAAGAAGGGCATCTCCAG SEQ ID NO: 190 hDNMT1_Adaptor_F ACACTCTTTCCCTACACGACGCTCTTCCGATCT AGTGTTCAGTCTCCGTGAACGTTC SEQ ID NO: 191 hDNMT1_Adaptor_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT TCCTTAGCAGCTTCCTCCTCCTT SEQ ID NO: 192 hCCR5_Adaptor_F ACACTCTTTCCCTACACGACGCTCTTCCGATCT AACAGTTTGCATTCATGGAGGGC SEQ ID NO: 193 hCCR5_Adaptor_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT AGTTTATCAGGATGAGGATGACC SEQ ID NO: 194 hFANCF_Adaptor_F ACACTCTTTCCCTACACGACGCTCTTCCGATCT CACTACCTACGTCAGCACCTGGGACC SEQ ID NO: 195 hFANCF_Adaptor_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT GGAAGTTCGCTAATCCCGGAACTGGA SEQ ID NO: 196 hGRIN2B_Adaptor_F ACACTCTTTCCCTACACGACGCTCTTCCGATCT CTCTCATTCTGCAGAGCAAATACCAGAGAT SEQ ID NO: 197 hGRIN2B_Adaptor_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTGCAAACACAAAGAAAGAGCATGTTAAA SEQ ID NO: 198 hEMX1_Adaptor_F ACACTCTTTCCCTACACGACGCTCTTCCGATCT GGCCTCCTGAGTTTCTCATCTGTGC SEQ ID NO: 199 hEMX1_Adaptor_R GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT CCTGCTTCGTGGCAATGCGCCAC SEQ ID NO: 200

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 μl 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.

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.

[Intensity of the cleaved fragment/total sum of the fragment intensity]×100 = DNA cleavage efficiency (%)

As a result, it was confirmed that approximately +10 DNA substitutions from the 3' end did not significantly affect the cleavage efficiency of the target gene sequence ( FIG. 2 ). However, it was confirmed that 12 or more consecutive DNA substitutions in the guide induced severe on-target cleavage efficiency. That is, it was confirmed that the 2'-OH sensitivity of the guide recognized by Cpf1 increased from the region far from the seed to the region closer to the seed. On the other hand, in the case of the target DNA cleavage efficiency when the pseudoknot structure at the 5' end, which is a region other than the proto-spacer, is sequentially changed by 4 nt from the end, the cleavage efficiency is higher in the chimeric DNA-RNA guide compared to the wild-type guide It was confirmed that it was significantly reduced (Fig.

Example 3. Analysis of genome cleavage efficiency according to DNA substitution of Cpf1 RNA guide seed portion

Following the target DNA cleavage experiment according to continuous DNA substitution, it was specifically confirmed which part of the target DNA and the 2'-OH of the seed region of the guide was recognized by the amino acid of Cpf1.

For two genes ( DNMT1, CCR5 ), genome cleavage efficiency was performed in the same manner as in Example 2 according to RNA guide single-nucleotide DNA substitution in the seed part close to the PAM (TTTN) sequence.

As a result, it was confirmed that the DNA cleavage efficiency was reduced at 7 to 10 parts from the PAM nucleotide sequence position (FIG. 4). That is, it was confirmed that the recognition of 2'-OH can be changed depending on the type of nucleotide sequence of the DNMT1 gene and the CCR5 gene.

On the other hand, as a result of substituting DNA for 4 bases in the RNA guide complementary to the portion close to the PAM sequence, it was confirmed that the cleavage efficiency was significantly lowered compared to the DNA substitution of a single base (FIG. 4).

Through the results of Examples 2 and 3, it was confirmed that the DNA substitution of the 3' region (up to about +10) in the guide compared to the DNA substitution of the seed or 5' region of the guide was effective in maintaining the cleavage activity of the target DNA. . Therefore, in the accuracy test for off-target cleavage efficiency, a guide in which the 3' region was substituted with DNA was used as a screening test group.

Example 4. Analysis of cleavage specificity by target gene using a chimeric DNA-RNA guide

In order to screen an effective guide according to DNA substitution, on-target and off-target cleavage efficiencies were measured in various genes ( DNMT1, CCR5, FANCF, GRIN2B, EMX1 ) to confirm off-target cleavage properties, respectively.

Specifically, 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 ).

As a result of measuring the cleavage efficiency for the DNMT1 target using the chimeric guides in Tables 2 to 4, in the case of DNA substitution at the 3' end of the guide, approximately 10 substitutions, specifically 8 substitutions, without on-target cleavage compensation (compensation) It was confirmed that the specificity increased until (Fig. 12A). In the case of single-nucleotide DNA substitutions in the seed region, changes from PAM up to 10 showed a tendency to decrease target DNA cleavage specificity, and in particular, successive changes within the seed region significantly increased on-target reward, resulting in off-target cleavage. It was confirmed that the contrast specificity showed a tendency to rapidly decrease (FIG. 12B).

When the CCR5 gene sequence was targeted, the on-target cleavage during the sequential 3' end DNA substitution of the guide was similar to that of the DNMT1 target, and it was confirmed that the specificity showed a similar trend because non-specific cleavage was not measured (Fig. 7). ). When the CCR5 gene was cut by applying a guide single-nucleotide DNA substitution in the seed region, specificity was reduced at 2, 4, 5, and 7th from PAM, and the rest was measured at a level similar to that of the wild-type RNA guide (FIG. 8) .

That is, it was confirmed that the continuous DNA substitution from the 3' end of the guide to the 8th consecutive DNA substitution had less on-target compensation and higher specificity than the DNA substitution of the continuous seed region. Therefore, the on/off cleavage ratio was confirmed by targeting more diverse gene sequences using the chimeric DNA-RNA guide in which the 3' end region was substituted ( FIGS. 9 to 11 and 14 ).

As a result of inducing in vitro cleavage, a similar result was confirmed in that the on/off cleavage rate was increased in the DNA substitution at the 3' end (up to about 8) of the guide on the nucleotide sequences of the other three genes (Fig. 14).

Through these results, it was confirmed that, in the case of DNA substitution at the 3' end of the guide, the specificity of target DNA cleavage was broadly improved without being greatly affected by the type of nucleotide sequence.

Example 5. Confirmation of restoration of intracellular genome editing efficiency by phosphorothioate improvement at the 3' end of the Cpf1 chimeric DNA-RNA guide

Upon 3' end DNA substitution of the chimeric DNA-RNA guide, there is a possibility that degradation of the chimeric DNA-RNA guide by the 3' end DNA exonuclease occurs.

Therefore, in order to improve the genome editing efficiency of the 3' end DNA substituted chimeric DNA-RNA guide, 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.

First, 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.

As a result, it was confirmed that cleavage activity similar to or better than that of wild-type RNA guide was induced even when the guide modified with PS was used at the 3' end (FIG. 16). On the other hand, it was confirmed that the non-target-specific cleavage was decreased and the specificity was relatively increased (FIG. 17).

Therefore, using a chimeric DNA-RNA guide (Table 4) modified with PS at the 3' end, indels were induced in the same gene sequence. Specifically, 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. After mixing with the Cpf1-chimeric guide premixed complex using 10 ⁵ cells, 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 μl 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).

In order to check whether genomic DNA has been genomically corrected, 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. for 25 minutes in a volume of 10 μl. did. After the reaction, the reaction was stopped by mixing a stop buffer (100 mM EDTA, 1.2% SDS), and DNA cleavage was checked by 2% agarose gel electrophoresis, and the calibration efficiency was measured using the ImageJ program.

In addition, 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 ℃, primer annealing: 30 sec at 58 ℃, elongation: 30 sec at 72 ℃, 35 cycles) was repeated to insert the adapter and index sequences at both ends of the amplicon (denaturation: 30 sec at 98 ℃) , primer annealing: 15 s at 62° C., elongation 15 s at 72° C., 35 cycles). Then, 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.

As a result, it was confirmed that the on-target compensation effect was largely recovered ( FIGS. 18 and 19 ), and it was confirmed that the effect could be enhanced through modification at the 3' end. In particular, it was confirmed that the Cpf1 gene scissors with improved target specificity by using the PS-modified chimeric DNA-RNA guide can be effectively operated in cells.

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

It was confirmed whether the chimeric DNA-RNA guide can be applied to Cpf1 derived from other microbial species other than AsCpf1.

Specifically, 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. In addition, 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.

As a result, a similar cleavage pattern appeared for the target nucleotide sequence and the non-target nucleotide sequence ( FIG. 20 ), and it was confirmed that the target specificity was also improved ( FIG. 21 ).

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

In order to confirm whether the DNA double helix is due to the form of the negative supercoil wound around the histone protein in the actual genome in the cell, the negative supercoil was removed and the genome editing efficiency was analyzed.

Specifically, 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).

Example 8. Comparison of target specificity of chimeric DNA-RNA guides

In the same manner as in the previous salpin method, DNMT1, GRIN2B, HPRT1 of AsCpf1 using a chimeric DNA-RNA guide (4nt at the 3' end of crRNA, 8nt length of continuous DNA substitution, +4, +8 is the number of substituted DNA) , RPL32P3 gene sequence target specificity was confirmed, and the results are shown in FIG. 23 .

23A to 23D show the results of confirming the target specificity of the DNMT1, GRIN2B, HPRT1, and RPL32P3 gene sequences, respectively.

As can be seen in FIG. 23, when using the chimeric DNA-RNA guide according to the present invention, excellent on-target efficiency was exhibited, and in particular, when 8 DNA sequences were included at the 3' end, the best effect was exhibited. .

Example 9. Investigation of target specificity for gene sequences on plasmids using chimeric DNA-RNA guides

In order to confirm the possibility of action in various forms of the system using the chimeric DNA-RNA guide according to the present invention, as shown in FIG. 24A , chimeric DNA-RNA-based AsCpf1 in higher animal cells (eg, HEK293FT) Upon delivery, an experiment was performed to compare the efficiency of target/non-target sequence correction on the plasmid.

Specifically, experiments on target/non-target sequence correction efficiency and target specificity for DNMT1 and GRIN2B were performed similarly to the above examples, and the results are shown in FIGS. 24B to 24E .

As can be seen from the figure, excellent proofreading efficiency and specificity were exhibited centered on the number of DNAs of approximately +8 for each of DNMT1 and GRIN2B.

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

As in FIG. 25A, an experiment was performed to confirm the genome editing efficiency by simultaneous delivery of chimeric DNA-chimeric DNA-RNA-based Cas12a and dead/nickase SpCas9 in higher animal cells (eg, HEK293FT). .

Specifically, as shown in FIG. 25A , an experiment was performed using the CCR56 target gene nucleotide sequence and in-silico predicted non-target nucleotide sequence information.

The experimental results are shown in B to C of FIG. 25 . 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). As can be seen from the above results, it was confirmed that excellent efficiency appeared when all of them were included.

In addition, the 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 . As can be seen in the figure, 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

Since HIV is a retrovirus, there is a high possibility of mutation of this protein, so it cannot be properly targeted, so there is no perfect treatment yet. Accordingly, by selectively removing the CCR5 gene in T cells using CRISPR-Cas12a (Cpf1) gene scissors, it was confirmed whether it is possible to use an effective gene therapy in the form of ex-vivo injection by autologous T cell correction in human subjects. .

Specifically, in order to purify a recombinant protein of AsCpf1 (Acidaminococcus sp. Cpf1), which structurally has well-defined interaction with guide RNA among known Cpf1 proteins, pET28a-Cas12a (AsCpf1) bacterial expression vector was transformed into E. coli BL21 (DE3) species. After conversion, it was incubated at 37˚C until OD=0.6. 48 hours after IPTG inoculation, bacterial cells were precipitated and the culture solution was removed, and the remaining precipitate was re-washed with bufferA [20mM Tris-HCl (pH8.0), 300mM NaCl, 10mM β-mercaptoethanol, 1% TritonX-100, 1mM PMSF] dissolved. Thereafter, the bacterial cell membrane was broken through sonication (ice, 3 min), and the cell lysate was harvested after centrifugation (5000 rpm, 10 min). First, 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. After bacterial cell precipitation, use 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 .

As can be seen in Figure 26, the 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, to obtain an activated gene scissors protein having a purity of 90% or more.

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.

Target gene location, type CRISPR-Cas12a (AsCpf1)
target sequence Guide RNA sequence (5'-3')
Among chimeric DNA-RNA, DNA is indicated in bold and underlined. hCCR5
(wt-crRNA) GTGGGCAACATGCTGGTCATCCTC TTTT (SEQ ID NO: 201) 5'AAUUUCUACUCUUGUAGAUGUGGGCAACAUGCUGGUCAUCCUC 3' (SEQ ID NO: 202) hCCR5 (chimeric DNA-RNA) GTGGGCAACATGCTGGTCATCCTC TTTT (SEQ ID NO: 203) 5' AAUUUCUACUCUUGUAGAUGUGGGCAACAUGCUGG TCATCCTC 3' ( SEQ ID NO: 204)

In Table 6 above, the PAM sequence (TTTN) in the target DNA gene is underlined, and the DNA sequence of the chimeric guide is shown in bold.

In addition, as can be seen in FIG. 27A , 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. In B, 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.

Based on the above information, a CCR5 gene targeting experiment of Cpf1 in animal cells using a chimeric DNA-RNA guide was performed. For the in vivo application of 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. In order to confirm the efficiency of target-specific genome editing using chimeric DNA-RNA in animal cells, the HEK293FT (ATCC) cell line was cultured. For the cell line, 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. For efficient delivery of the gene scissors vector into the nucleus, electroporation (Lonza, V4XC-2032)) was used, and As/LbCp1 (500 ng), nCas9 (D10A (100 ng)), crRNA (150 pmol), sgRNA (15 pmol) of vector and guide RNA were delivered. After 12 hours of electroporation, the same amount of crRNA and sgRNA was delivered into the cell using 2.8 ul of Lipofectamine 3000 (ThermoFisher) and 2.0 μl of P3000 reagent. RNA transfer was carried out. After electroporation, 72 hours later, the genomic DNA of the HEK293FT cell line was extracted. Using the extracted genomic DNA, the target gene and the predicted off-target gene location were analyzed through targeted amplicon sequencing. The saved Fastq file was analyzed with Cas-Analyzer code and the calibration efficiency (%) was calculated.

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.

Specifically, as a result of targeting a specific nucleotide sequence (table) in the CCR5 gene in a human cell line (HEK293FT) using Cpf1 gene scissors, 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).

In addition, as a result of targeting a specific nucleotide sequence (table) in the CCR5 gene in a human cell line (HEK293FT) using AsCpf1 gene scissors, 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).

Even when the same experiment was targeted using LbCpf1 gene scissors, it was confirmed that the target specificity increased more than twice when +8DNA crRNA in which the 3' end of the guide RNA was replaced with DNA compared to WT crRNA was used (FIG. 29C).

Example 12. Application of gene therapy for oncogene-targeted therapy

Genes with origin that can cause cancer progression in the human body are known as oncogenes. When 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.

Specifically, as shown in FIG. 30 , 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.

Target gene location, type CRISPR-Cas12a (AsCpf1)
target sequence Guide RNA sequence (5'-3')
Among chimeric DNA-RNAs, DNA is in bold and underlined. hBRAF (wt- crRNA) TTTTGGTCTAGCTACAGAGAAATCTCGA TTTT (SEQ ID NO: 205) 5' AAUUUCUACUCUUGUAGAUGGUCUAGCUACAGAGAAAUUCUCGA 3' (SEQ ID NO: 206) hBRAF (chimeric DNA-RNA) TTTTGGTCTAGCTACAGAGAAATCTCGA TTTT (SEQ ID NO: 207) 5' AAUUUCUACUCUUGUAGAUGGUCUAGCUACAGAGA AATCTCGA 3' (SEQ ID NO: 208)

In Table 7 above, the PAM sequence (TTTN) in the target DNA gene is underlined, and the DNA sequence of the chimeric guide is shown in bold.

In FIG. 31, the Cpf1 target nucleotide sequence for target-specific removal of the oncogenic BRAF mutant gene was specifically described. As can be seen in FIG. 31A , 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): The mutation from the normal gene (1799T) to the oncogenic gene (1799T>A) was specifically described. In addition, as can be seen in FIG. 31B , a point mutation in the BRAF gene (underlined T). MT: mutant gene (oncogene), WT: normal gene (proto-oncogene) were described.

An in-vitro level oncogene-specific cleavage experiment was performed.

In order to compare and analyze the cleavage efficiency of Cpf1 using wild-type and chimeric DNA-RNA guides (D+8) for the normal BRAF gene (1799T) and the oncogenic gene BRAF (1799T>A), 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. Then, hBRAF_Mutation_amplicon 2㎍, hBRAF_wild-type_amplicon 2㎍, AsCpf1 or LbCpf1 protein 2.8㎍, crRNA (WT) 600ng or chimeric DNA-RNA (D+8) 600ng cleavage buffer (NEB3.1 buffer, deionized water up to 10㎍) 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 results are shown in FIGS. 32 and 33 .

In order to confirm the target specificity of the oncogene BRAF (1799T>A) compared to the normal BRAF gene (1799T), wild-type and chimeric DNA for the oncogene BRAF (1799T>A) and the normal BRAF gene (1799T) as shown in FIG. 32 The cleavage efficiency of Cpf1 using -RNA guide (D+8) was comparatively analyzed.

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 ).

Example 13. Application of gene therapy for targeted treatment of degenerative eye diseases

In order to develop a treatment that fundamentally and locally inhibits angiogenesis at the DNA level using the CRISPR-Cpf1 gene scissors with improved target specificity, 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 .

Target gene location, type CRISPR-Cas12a (AsCpf1)
target sequence Guide RNA sequence (5'-3')
Among chimeric DNA-RNAs, DNA is in bold and underlined. 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)

In Table 8 above, the PAM sequence (TTTN) in the target DNA gene is underlined, and the DNA sequence of the chimeric guide is shown in bold.

In addition, the Cpf1 target nucleotide sequence for target-specific removal of the VEGFA gene is shown in FIG. 34 . 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. 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.

An in-vitro level VEGFA gene-specific cleavage experiment was performed.

In order to compare and analyze the cleavage efficiency of Cpf1 using wild-type and chimeric DNA-RNA guide (D+8) for hVEGFA target sequence (on-target) and off-target sequence (off-target), respectively In vitro cleavage assay was performed. Each nucleotide sequence of the hVEGFA target gene (on-target) and off-target gene (off-target) was synthesized and inserted into the T-vector, and PCR was performed on each containing the target nucleotide sequence to obtain an amplicon. Then, 600ng of hVEGFA_on-target amplicon, 600ng of hVEGFA_off-target amplicon, 2.8㎍ of AsCpf1-FLAG protein, 600ng of crRNA (WT), 600ng of crRNA (D+8) were mixed with cleavage buffer (NEB3.1 buffer, DeIonized Water up to 10 μl) and After mixing and incubating for 1 hour in an incubator at 37°C, the band pattern was confirmed by electrophoresis on agarose 2% gel at 200V-20min. In addition, the DNA cleavage efficiency (cleavage efficiency = cleaved band intensity/total intensity X100) was calculated using ImageJ software.

In order to confirm the non-target specificity for the target VEGFA gene, 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.

The DNA cleavage efficiency of Cpf1 gene scissors using wild-type and chimeric DNA-RNA guides (D+8) for on-target sequences in the target gene was similar, and non-target sequences (off-target sequences) were similar. target), when the chimeric DNA-RNA guide (D+8) was used, the DNA cleavage efficiency was significantly reduced (FIG. 36 A), so the Cpf1 gene scissors using the chimeric DNA-RNA guide (D+8) was not It was shown that the specificity of the on-target sequence in the target gene compared to the target sequence (off-target) was increased ( FIG. 36B ).

Up to now, the present invention has been looked at focusing on the embodiments thereof. Those of ordinary skill in the art to which the present invention pertains will understand that the present invention can be implemented in a modified form without departing from the essential characteristics of the present invention. Therefore, the disclosed embodiments are to be considered in an illustrative rather than a restrictive sense. The scope of the present invention is indicated in the claims rather than the foregoing description, and all differences within an equivalent scope should be construed as being included in the present invention.

Claims (20)

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 DNA encoding the same A composition for genome editing comprising a. The method according to claim 1, wherein the guide is modified with a phosphate whose 3'-terminus is phosphonate, phosphorothioate or phosphotriester; modified by biotinylation; or LNA, HNA, CeNA, 2'-methoxyethyl, 2'-O-alkyl, 2'-O-allyl, 2'-C-allyl, 2'-fluoro, 2'-deoxy, 2'- A composition for genome editing that is modified by any one selected from the group consisting of hydroxyl and combinations thereof. The composition for genome editing according to claim 1, wherein the DNA of the chimeric DNA-RNA guide is substituted at the 3' end of the guide. The composition for genome editing according to claim 3, wherein the number of DNA is 6 to 10. The composition of claim 1, wherein the composition further comprises SpCas9 nickase (D10A) or inactive (Dead) SpCas9 nickase (D10A or H840A). A method for producing a transformant comprising the step of introducing the composition for genome editing of claim 1 into an isolated cell or organism. inactive Cpf1 (dCpf1) 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 DNA encoding the same A composition for inhibiting gene expression comprising a. The composition for inhibiting gene expression according to claim 7, wherein the guide has a 3'-end modified with phosphorothioate. The composition for inhibiting gene expression according to claim 7, wherein the DNA of the chimeric DNA-RNA guide is substituted at the 3' end of the guide. The composition for inhibiting gene expression according to claim 7, wherein the composition further comprises SpCas9 nickase (D10A). 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 pharmaceutical composition comprising a DNA encoding the same. 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 genetic disease, non-hereditary disease, viral infection, bacterial infection, cancer, or autologous disease comprising DNA encoding the same A pharmaceutical composition for the prevention or treatment of immune diseases. Cpf1 protein or DNA encoding the same; And 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, A method of altering expression of a gene product, comprising introducing into a cell that contains and expresses a DNA molecule having a target sequence and encoding the gene product. Cpf1 protein or DNA encoding the same; And 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, preventing a disease associated with a mutation or single-nucleotide polymorphism (SNP) in a subject by altering the expression of the gene product, comprising introducing into a cell that contains and expresses a DNA molecule having a target sequence and encoding the gene product; How to improve or treat. 15. The method of claim 14, wherein the disease associated with the mutation or single-nucleotide polymorphism (SNP) is a genetic disease, a non-genetic disease, a viral infection, a bacterial infection, cancer, or an autoimmune disease. A method of preventing, ameliorating or treating a disease associated with a form (SNP). For use in genome editing, Cpf1 protein or DNA encoding the same; and A composition comprising a chimeric DNA-RNA guide comprising a nucleotide sequence capable of hybridizing with a target nucleotide sequence or a DNA encoding the same. Cpf1 protein or DNA encoding the same for use in the prevention or treatment of diseases associated with mutations or single-nucleotide polymorphisms (SNPs); 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. 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 DNA encoding the same for genome editing. In the manufacture of a medicament for use in the prophylaxis 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 capable of hybridizing with a target nucleotide sequence or a composition comprising DNA encoding the same. Cpf1 protein or DNA encoding the same; and A chimeric DNA-RNA guide comprising a target nucleotide sequence and a hybridizable nucleotide sequence, or a gene diagnosis composition comprising a DNA encoding the same.

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