KR102551876B1 - Composition for Genome Editing or Inhibiting Gene Expression comprising Cpf1 and Chimeric DNA-RNA Guide - Google Patents

Abstract

The present invention relates to a composition for genome editing or expression inhibition comprising Cpf1 and a chimeric DNA-RNA guide, which has a similar indel efficiency to that of a conventional RNA guide, but superior target specificity to an RNA guide, thus providing stability and high therapeutic effect. It can be effectively applied to gene therapy that requires

Description

Composition for Genome Editing or Inhibiting Gene Expression 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 referred to as Cpf1) is a CRISPR system belonging to class II, type V. Like the Cas9 gene scissors, it consists of a bi-lobed structure of a nuclease domain and a recognition domain, and is a single-stranded crRNA. It is known to bind to a target DNA double helix using a DNA-RNA hybrid duplex. Since the discovery of Cas9, Cpf1 has been attracting attention as an excellent genetic scissors to complement the restrictive parts of PAM. In addition, Cpf1 gene scissors specifically induce DNA double helix cleavage by recognizing the thymidine (T)-rich portion as PAM (TTTN or TTN) in addition to the guanine (G)-rich region of the intracellular locus. Because of this, it is also widely used as target-specific genetic scissors. Most recently, there is a trend to improve the possibility as a gene therapy by engineering the Cpf1 protein to expand the range of genes that can be targeted by expanding the PAM, or by increasing the efficiency of DNA double helix cleavage through modification of the guide RNA.

To date, it is known that Cpf1 structurally recognizes a protospacer of 24 bases, and the internal seed region is approximately 5 to 10 bases from the PAM sequence. In the process of recognizing target DNA, it is more sensitive to mismatches than CRISPR-Cas9, and when a mismatch is introduced into the seed part of the protospacer region, its activity is significantly lowered, and it has been known as a genetic scissors with excellent target specificity. . However, since the possibility of cleavage for mismatches other than the seed part still exists, in the DNA cleavage process including the target sequence of Cpf1 with increased activity later, such off-target cleavage Non-detection issues may be further highlighted.

Accordingly, the present inventors completed the present invention by conducting research to develop CRISPR-Cpf1 gene scissors with excellent target specificity and improved indel efficiency.

Korean Patent Publication No. 10-2018-0028996

One object of the present invention is a Cpf1 protein or DNA encoding the same; and 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 a separated cell or organism.

Another object of the present invention is to provide a method for altering the expression of a gene product, including 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 it; and 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 change 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 a gene product (wherein the DNA molecule is It is to provide a method for preventing, improving or treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) in a subject by including a mutant sequence).

Another object of the present invention is a Cpf1 protein or DNA encoding the same; And to provide a pharmaceutical composition 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 a Cpf1 protein or DNA encoding the same; And to provide a pharmaceutical use using a pharmaceutical composition 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 a Cpf1 protein or DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a composition for genetic diagnosis comprising a DNA encoding the same.

One aspect of the present invention is a Cpf1 protein or DNA encoding the same; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

“Cpf1” is a type V CRISPR system protein. It is similar to Cas9, a type II CRISPR system protein, in that a single protein binds to crRNA to cleave a target gene, but there is a big difference in how it works. In particular, since the Cpf1 protein operates as a single crRNA, there is no need to use both crRNA and trans-activating crRNA (tracrRNA) at the same time as in the case of Cas9, or to artificially create a single guide RNA (sgRNA) combining tracrRNA and crRNA. In addition, in the Cpf1 system, unlike Cas9, the PAM is present at the 5' position of the target sequence, and the length of the guide RNA determining the target is shorter than that of Cas9. Utilizing these features, Cpf1 has the advantage that genome editing is possible even for target sequences in which Cas9 cannot be used, and that it is relatively easy compared to Cas9 for preparing crRNA, a guide RNA. In addition, since Cpf1 generates a 5' overhang (sticky end) rather than a blunt-end at the site where the target DNA was cut, more accurate and diverse gene editing is possible. In the present specification, a technology for correcting a target genome more conveniently, accurately and effectively using the Cpf1 system is provided.

For example, the Cpf1 protein is Candidatus genus, Lachnospira genus, Butyrivibrio genus, Peregrinibacteria genus, Acidominococcus genus, Porphyromonas ( Porphyromonas genus, Prevotella genus, Francisella genus, Candidatus Methanoplasma genus, 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, and Eubacterium eligens . In one example, 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 eligen be from s It may be, but is not limited thereto.

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

Cpf1 protein origin microorganism 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 Methanoplasma termitum 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 or synthetic method. The Cpf1 protein may additionally include elements commonly used for delivery into the nucleus of eukaryotic cells (eg, a 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 an element commonly used for delivery into the nucleus of eukaryotic cells (eg, a nuclear localization signal (NLS; eg, PKKKRKV, KRPAATKKAGQAKKKK, or a nucleic acid molecule encoding the same)), etc. ) to the N-terminus or C-terminus (or 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 can be used in the form of a purified protein, a nucleic acid encoding it (DNA/RNA), or a recombinant vector containing the DNA.

The Cpf1 protein can be linked with a tag that is advantageous for isolation and/or purification. For example, a small peptide tag such as a His tag, a Flag tag, an S tag, or a Glutathione S-transferase (GST) tag or a Maltose binding protein (MBP) tag 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 the microorganism derived therefrom.

As used herein, the term 'genome editing' refers to a nucleic acid molecule (one or more, e.g., 1-100,000bp, 1-10,000bp, 1 -1000, 1-100 bp, 1-70 bp, 1-50 bp, 1-30 bp, 1-20 bp, or 1-10 bp) loss, alteration, and/or recovery (modification) of gene function by deletion, insertion, substitution It can be used to mean to do.

As used herein, the term "chimeric DNA-RNA guide" refers to a part of the RNA of crRNA (crispr RNA) specific to the target DNA substituted with DNA, and the chimeric DNA-RNA guide complexes with the Cpf1 protein. can form and bring the Cpf1 protein to the target DNA. DNA substitution of the guide RNA can reduce off-target DNA sequence cleavage by changing the binding energy of the guide and target DNA. In addition, it can be synthesized inexpensively, and it can be freed from the issue of 5' phosphate, which inevitably occurs during laboratory self-production, and can avoid induction of immune responses. It can be used more safely when Cpf1 is introduced. In addition, compared to RNA that is chemically unstable in an aqueous solution, DNA-type guides have the advantage of being easy to chemically modify and easy to apply.

In such a chimeric DNA-RNA guide, the DNA may be substituted at the 3' end of the guide. Specifically, the DNA preferably has 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 a chimeric DNA-RNA guide in which 6 to 10 RNAs from the 3' end of the crRNA are substituted with DNA exhibits high specificity and cleavage activity, the substitution of the crRNA is preferably performed at the 3' end of the crRNA.

Since a chimeric DNA-RNA guide in which 6 to 10 RNAs from the 3' end of the crRNA are substituted with DNA exhibits high specificity and cleavage activity, the substitution of the crRNA is preferably performed 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 bonds between bases of nucleotide residues.

In the present specification, a nucleotide sequence capable of hybridizing with a gene target site is 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more of the nucleotide sequence (target sequence) of the gene target site. Refers to a nucleotide sequence having a sequence complementarity of at least, or 100% (hereinafter, unless otherwise specified, the same meaning is used, and the sequence homology can be confirmed using a conventional sequence comparison tool (such as BLAST)) .

The gene editing composition of the present invention is 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 encoding It can be introduced into cells or organisms in the form of a recombinant vector containing nucleotides that contain Cpf1 protein and chimeric DNA-RNA, or in the form of ribonucleic acid proteins forming a complex thereof.

The chimeric DNA-RNA guide of the present invention can hybridize with a target DNA.

The gene editing composition of the present invention can be applied to eukaryotic organisms. The eukaryotic organism is a eukaryotic cell (eg, a fungus such as yeast, a eukaryotic animal and/or a eukaryotic plant-derived cell (eg, an embryonic cell, a stem cell, a somatic cell, a germ cell, etc.)), a eukaryotic animal (eg, For example, vertebrates or invertebrates, more specifically, humans, primates such as monkeys, mammals including dogs, pigs, cows, sheep, goats, mice, rats, etc.), and eukaryotic plants (eg, green algae, etc.) It may be selected from the group consisting of algae, monocotyledonous or dicotyledonous plants such as corn, soybean, wheat, rice, etc.), but is not limited thereto.

The gene editing composition of the present invention is 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 encoding 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 a polymerase and initiating transcription of a downstream (3' direction) coding or non-coding sequence.

As used herein, the term "operably linked" means 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, and a transcription termination sequence.

The promoter of the present invention is one of the transcription control sequences that control the initiation of transcription of a specific gene, and may be a polynucleotide fragment of about 100 bp to about 2500 bp in length.

The promoter can be used without limitation as long as it can regulate transcription initiation in cells, for example, eukaryotic cells (eg, plant cells, or animal cells (eg, mammalian cells such as humans and mice), etc.) do. For example, the promoter is a CMV promoter (cytomegalovirus promoter; eg, 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, tk promoter of HSV, SV40E1 promoter, respiratory syncytial virus (Respiratory syncytial virus (RSV) promoter, metallothionein 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 species selected from the group consisting of a human granulocyte-macrophage colony stimulating factor (GM-CSF) gene promoter, etc., but is not limited thereto. In one example, the promoter may be selected from the group consisting of a CMV immediate-early promoter, a U6 promoter, an elongation factor 1-a (EF1-alpha) promoter, and an EF1-alpha short (EFS) promoter. The transcription termination sequence may be a polyadenylation sequence (pA) or the like. The origin of replication may be the f1 origin of replication, the SV40 origin of replication, the pMB1 origin of replication, the adeno origin of replication, the AAV origin of replication, or the BBV origin of replication.

The vector of the present invention may be selected from the group consisting of 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 are 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 recombinant expression vector of the present invention can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cutting and linking can be performed using enzymes generally known in the art. there is.

According to one embodiment of the present invention, the 3'-end of the guide may be modified. By modifying the 3'-end of the chimeric DNA-RNA guide of the present invention, it can exhibit high target specificity and excellent indel efficiency without being affected by DNA exonuclease in cells.

Specifically, it may include modification to phosphate, which 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' - It may be 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 being affected by 3' DNA exonuclease in cells by modifying the 3'-terminus with phosphorothioate. there is.

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 number of DNAs may be 6 to 10.

In the case of chimeric DNA-RNA guides in which RNA at the 5' end of crRNA or seed region is substituted with DNA, the cleavage activity is low. On the other hand, since a chimeric DNA-RNA guide in which 6 to 10 RNAs from the 3' end of the crRNA are substituted with DNA exhibits high specificity and cleavage activity, the substitution of the crRNA is preferably performed 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 the S. pyogenes Cas9 nickase with the D10A mutation. Inactive SpCas9 (dead SpCas9) (D10A, or H840A) refers to S. pyogenes Cas9 having D10A, H840A mutations.

SpCas9 nickase (D10A) can remove the negative supercoil of the DNA double helix present in cells, so it can improve the genome editing efficiency of chimeric DNA-RNA guides in cells.

Another aspect of the present invention provides a method for producing a transformant comprising introducing the composition for genome editing into a separated cell or organism. Here, the organism may exclude humans.

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 known in the art, it is suitable for host cells. This can be done with a selection of standard techniques. These methods include, for example, electroporation, calcium phosphate (CaPO ₄ ) precipitation, calcium chloride (CaCl ₂ ) precipitation, microinjection, polyethylene glycol (PEG) method, DEAE-dextran method, cationic A liposome method, a lithium acetate-DMSO method, and the like 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, including 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). Reference).Methods using selectable markers that can be visualized by fluorescence can be further sorted using fluorescence activated cell sorting (FACS) technology.The isolated engineered cells can be used to establish cell lines for transplantation. The isolated altered cells may be cultured using any suitable method to produce a stable cell line.

Another object of the present invention is to change 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 a gene product, so that a mutation or single mutation in a subject - To provide a method for preventing, improving or treating diseases associated with nucleotide polymorphisms (SNPs). wherein the DNA molecule contains a mutant or SNP mutant sequence.

A DNA molecule can contain at least one, two, three, four or more SNPs or mutation sites, and the methods described herein can be used to generate gene products associated with at least one, two, three, four or more SNPs or mutation sites. alter expression. That is, it is possible to alter the mutant sequence at multiple SNP sites or ancestral 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-genetic diseases, viral infections, bacterial infections, cancers, or autoimmune diseases. do.

As used 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. An abnormality may be a mutation, insertion or deletion. An abnormality may be a mutation, an insertion or a deletion. Can affect the coding sequence of a gene or its regulatory sequence Genetic 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, xeroderma pigmentosum, Fanconi's anemia, retinitis pigmentosa, telangiectasia, bloom syndrome (Bloom's syndrome), retinoblastoma and Tay-Sachs disease, but are not limited thereto.

The non-genetic disease target is to treat a disease by regulating a normal gene other than a mutated gene using Cpf1, and may typically be age-related macular degeneration (AMD), but is not limited thereto.

Here, viruses, bacterial infections, or diseases caused by these include, but are not limited to, AIDS, avian influenza, influenza, CMV infectious diseases, 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, colon cancer, stomach cancer, tongue cancer, skin cancer, brain tumor, uterine cancer, head or It includes, but is not limited to, 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.

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 platelet reduced purpura, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture syndrome, bullous pemphigoid, discoid lupus erythematosus, ulcerative colitis, or dense deposits; and the like.

In the context above, treatment provides a positive therapeutic response to a disease or condition. By “positive therapeutic response” is intended an amelioration of a disease or condition and/or an amelioration of symptoms associated with a disease or condition.

Improvement of the symptoms includes administration of an effective amount or a therapeutically effective amount of the composition for genome editing. "Effective amount" or "therapeutically effective amount" refers to an amount of an agent sufficient to produce a beneficial or desired result. A therapeutically effective amount may vary depending on one or more of the following: the subject and 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 DNA encoding the same; And to provide a pharmaceutical composition comprising a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

The dosage form of the pharmaceutical composition of the present invention may be for parenteral use. When formulated, 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 sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.

The pharmaceutical composition of the present invention can be administered parenterally, intratumorally, intravenously, intramuscularly, intracutaneously, subcutaneously, intraperitoneally, intraarterially, intraventricularly, intralesionally, intrathecally, topically, 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 in its range depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate and severity of the disease, and can be appropriately selected by those skilled in the art. can For desirable effects, the pharmaceutical composition of the present invention may be administered at 0.01 ug/kg to 100 mg/kg per day, specifically at 1 ug/kg to 1 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. Accordingly, the dosage is not intended to limit the scope of the present invention in any way.

Another object of the present invention is a Cpf1 protein or DNA encoding the same; And to provide a pharmaceutical use using a pharmaceutical composition comprising a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

The medicinal use herein may be for preventing, ameliorating or treating a disease associated with a mutation or single-nucleotide polymorphism (SNP) in a subject. More specifically, it is intended to prevent, ameliorate or treat diseases associated with mutations or single-nucleotide polymorphisms (SNPs), including genetic diseases, non-genetic diseases, viral infections, bacterial infections, cancers, or autoimmune diseases. can

Another object of the present invention is a Cpf1 protein or DNA encoding it for use in genome editing; and 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 use it for 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 it; and 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 be used to target and detect mutations in specific DNA or RNA of cancer, genetic diseases, or infection by viruses. Cpf1 protein or DNA encoding it in accurately distinguishing between normal and mutant genes; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

The gene diagnosis composition 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 DNA encoding the same; and

It is to provide a genome editing use of a composition 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 prepare a medicament for use in the prevention or treatment of diseases associated with mutations or single-nucleotide polymorphisms (SNPs),

Cpf1 protein or DNA encoding it; And to provide a use of a composition comprising a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

Another aspect of the present invention is an inactive Cpf1 (dCpf1) protein or DNA encoding it; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same.

In the composition for inhibiting gene expression of the present invention, parts overlapping with the above may be used in the same meaning as the above.

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 can be suppressed or reduced, so it is useful for regulating 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 known in the art to remove or reduce the activity of the Cpf1 protein.

According to one embodiment of the present invention, the 3'-end of the guide may be 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 the composition for genome editing or expression suppression including Cpf1 and chimeric DNA-RNA guide, the indel efficiency is similar to that of the existing RNA guide, but the target specificity is superior to that of the RNA guide, so genes requiring stability and high therapeutic effect It can be applied effectively to treatment, etc.

1 is a schematic diagram showing the interaction between crRNA and target DNA single strands in AsCpf1 protein. In order to confirm the performance of partial DNA substitution of crRNA, the 3' end, 5' end, or seed region were substituted with DNA. The RNA part of crRNA is blue and the DNA part is red (underlined) (numbers are number of substituted DNAs).
Figure 2 shows the target gene of AsCpf1 ( DNMT1 : Orange, CCR5 : Green) It is a graph showing the cutting efficiency. Cr1, Cr2, Cr3, Cr4, Cr5, Cr6, Cr7 and Cr8 respectively represent the chimeric guide sequences of crRNA1, crRNA2, crRNA3, crRNA4, crRNA5, crRNA6, crRNA7 and crRNA8 corresponding to Table 2, and CrS1, CrS2, CrS3, CrS4, CrS5, CrS6, CrS7 and CrS8 represent chimeric guide sequences of crRNA51, crRNA52, crRNA53, crRNA54, crRNA55, crRNA56, crRNA57 and crRNA58 corresponding to Table 3, respectively.
Figure 3 is a graph showing the cleavage efficiency of AsCpf1's target gene ( DNMT1 : target (orange, CCR5 : target (green))) using a chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 5' end of crRNA).
4 shows that the target gene ( DNMT1 : orange, CCR5 : Green) is a graph showing the cutting efficiency.
Figure 5 shows the target gene ( DNMT1 : target (orange), off-target (blue, gray, yellow) for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 3' end of crRNA) )) It is a graph showing the cutting efficiency.
Figure 6 shows target gene ( DNMT1 : target (orange), off-target (blue, gray, yellow)) cleavage to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (replacing the seed region of crRNA with DNA) Here is a graph showing the efficiency.
Figure 7 is a target gene ( CCR5 : target (green), off-target (orange, gray)) for confirming the target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution in units of 4 bp from the 3' end of crRNA) It is a graph showing the cutting efficiency.
Figure 8 shows the cleavage efficiency of the target gene ( CCR5 : target (green), off-target (orange, gray)) to confirm the target specificity of AsCpf1 using a chimeric DNA-RNA guide (replacing the seed region of crRNA with DNA) is the graph shown.
9 is a graph showing the cleavage efficiency of target genes and off-target genes ( FANCF : target (blue), off-target (orange, gray)) using a chimeric DNA-RNA guide (3' end 4bp, 8bp DNA replacement of crRNA) .
10 is a graph showing the cleavage efficiency of target genes and off-target genes ( GRIN2B : target (blue), off-target (orange, gray)) using a chimeric DNA-RNA guide (3' end 4bp, 8bp DNA replacement of crRNA) am.
11 is a graph showing the cleavage efficiency of target genes and off-target genes ( EMX1 : target (blue), off-target (orange, gray)) using a chimeric DNA-RNA guide (3' end 4bp, 8bp DNA replacement of crRNA) .
12 shows (A) DNMT1 target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA substitution in units of 4 bp from the 3' end of crRNA, numbers indicate the number of substituted DNA) and (B) chimeric DNA-RNA guide ( It is a graph showing the DNMT1 target specificity of AsCpf1 using crRNA seed region DNA replacement).
13 shows (A) CCR5 target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 3' end of crRNA) and (B) chimeric DNA-RNA guide (DNA replacement in the seed region of crRNA) It is a graph showing the CCR5 target specificity of AsCpf1 using .
14 is a graph showing (A) FANCF, (B) GRIN2B, and (C) EMX1 target specificity of AsCpf1 using a chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 3' end of crRNA).
15 is a diagram showing a gene editing pattern using a chimeric DNA-RNA guide.
Figure 16 shows the DNMT1 gene and off-target cleavage efficiency using a chimeric DNA-RNA guide (3' end phosphorothioate (PS) improvement based on DNA substitution in units of 4 bp from the 3' end of crRNA). it's a graph
17 is a graph showing the target specificity of a chimeric DNA-RNA guide whose 3' end is modified with PS.
18 is a diagram showing results of intracellular DNMT1 gene correction using a 3' end PS modified chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 5' end of crRNA).
19 is a diagram showing the efficiency of intracellular DNMT1 gene editing using a 3' end PS improved chimeric DNA-RNA guide (DNA replacement in units of 4 bp from the 5' end of crRNA). 4T indicates extension of 4 thymine bases to avoid inhibiting the activity compared to direct modification of PS at the 3' end of the chimeric guide. 4T-PS indicates modification with PS by extending four thymidine bases at the 3' end of the chimeric guide.
20 is a graph comparing the cleavage efficiencies of target sequences in the DNMT1 gene of AsCpf1, LbCpf1, and FnCpf1 gene scissors based on chimeric DNA-RNA guides using DNA amplicons. 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, and CrRNA3 in Table 2, respectively. -4, CrRNA4, CrRNA5, CrRNA6, CrRNA7 and CrRNA8.
21 is a graph showing the cutting efficiency of DNMT1 target gene and off-target gene by FnCpf1 gene scissors based on chimeric DNA-RNA guides.
22 is a diagram showing a nick created by SpCas9 nickase and a double helix cleavage site induced by CRISPR-Cpf1.
23 shows chimeric DNA-RNA guides (contiguous DNA substitutions of 4nt and 8nt lengths at the 3' end of crRNA, +4 and +8 for DNMT1, GRIN2B, HPRT1, and RPL32P3 (A, B, C, and D, respectively) The result of comparing the target specificity of the AsCpf1 gene sequence using the number of substituted DNAs) is shown (On/Off1 ratio (specificity)).
24 shows the results of target specificity investigation for gene sequences on plasmids using chimeric DNA-RNA guides. Figure 24A shows the results of comparison of target/non-target sequence editing efficiency on plasmid when chimeric DNA-RNA-based AsCpf1 delivery in higher animal cells (HEK293FT), and Figure 24B shows target for DNMT1 sequence on plasmid / Shows the results of comparison of off-target sequence editing efficiency, C in FIG. 24 shows the comparison result of target specificity for DNMT1 nucleotide sequence by next-generation sequencing analysis, and D in FIG. 24 shows target / ratio for GRIN2B nucleotide sequence on plasmid 24E shows the target specificity comparison result for the GRIN2B nucleotide sequence by next-generation sequencing analysis.
Figure 25 shows the results of investigation of Cas12a (Cpf1) gene editing activity by the combined use of the CCR5 gene target 3' end PS (phosphorothioate) improved chimeric guide and the nickase SpCas9. 25A shows genome editing by co-delivery of chimeric DNA-RNA-based Cas12a and dead/nickase SpCas9 in HEK293FT cells (Table: CCR5 target gene sequence and in-silico predicted off-target sequence information). 25B shows the results of NGS genome editing efficiency analysis by co-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), C in FIG. 25 shows the increase in genome editing specificity by the dead/nickase SpCas9 complex treatment.
26 is a result of purification of the gene scissors CRISPR-Cas12a (Cpf1) recombinant protein using Affinity column (Ni-NTA resin). AsCpf1 ( Acidaminococcus sp. Cpf1) and LbCpf1 (Cpf1), each of which has a (6X)His-tag attached to the N-terminus, were isolated/purified in bacterial cells to obtain activated gene scissors proteins with a purity of 90% or more. indicate
27A is a Cpf1 target nucleotide sequence for target-specific deletion of the CCR5 gene. The red color indicates the PAM (TTTN) nucleotide sequence, and the yellow color indicates the target nucleotide sequence, and B in FIG. The part where mismatch with the guide RNA in the base sequence (off-target) is shown in red).
Figure 28 shows the results of improving the CCR5 gene targeting specificity compared to non-targeting in animal cells (HEK293FT) of AsCpf1 using chimeric DNA-RNA guide (NC: negative control, control group not treated with genetic scissors, Only Cas12a: Cas12a protein Control group treated only, WT crRNA: treated with Cas12a using a previously used RNA-type guide, +8DNA crRNA: treated with Cas12a in which 8 nt at the 3' end of the existing RNA-type guide was substituted with DNA )
Figure 29 shows the enhancement of CCR5 gene targeting specificity compared to non-targeting in animal cells (HEK293FT) of LbCpf1 using a chimeric DNA-RNA guide (NC: negative control, control group not treated with genetic scissors, Only Cas12a: only Cas12a protein Treated control group, WT crRNA: Cas12a using a previously used RNA-type guide, +8DNA crRNA: Cas12a in which 8 nt at the 3' end of the existing RNA-type guide was substituted with DNA) .
30 shows the principle of target-specific oncogene removal. By applying Cpf1 gene scissors with improved target specificity that can cut only the oncogene BRAF (1799T>A) without cutting the normal gene BRAF, the oncogene that is the source of cancer cells is removed, thereby reducing cancer cells. It shows a schematic diagram of killing.
31 shows the Cpf1 target nucleotide sequence for target-specific removal of oncogenic BRAF mutant gene. 31A is orange: 24nt nucleotide sequence target of CRISPR-Cpf1 gene scissors, blue: PAM (TTTN) sequence required for target recognition by CRISPR-Cpf1 gene scissors, red: oncogenic gene within normal gene (1799T) (1799T>A) indicates a mutation, and B in 31 indicates a point mutation (MT: oncogene, WT: proto-oncogene) in the BRAF gene.
Figure 32 shows the specificity of the oncogenic BRAF mutation gene compared to the normal gene of Cpf1 using a chimeric DNA-RNA guide (NC: negative control, control group not treated with genetic scissors, WT: previously used RNA-type guide , D+8: Process Cas12a by substituting DNA for 8 nt at the 3' end of the existing RNA guide, Asterisk: Show truncated DNA BRAF MT: Oncogene BRAF (1799T>A ), BRAF WT: normal gene BRAF (1799T)
33 shows the enhancement of oncogene target specificity of Cpf1 compared to a normal gene using a chimeric DNA-RNA guide. Figure 33A shows the cleavage comparison experiment of existing wt-crRNA and chimeric DNA-RNA substituted with 8 DNAs for the normal gene BRAF (1799T) and oncogene BRAF (1799T>A), and Figure 33B shows A The value calculated from is 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 experimental group not treated with genetic scissors, WT-crRNA: treated with Cas12a using a previously used RNA-type guide, D+8-crRNA: 3' end of the existing RNA-type guide treated with Cas12a in which 8 nt were substituted with DNA BRAF MT/WT ratio: Cleavage target specificity for oncogene BRAF (1799T>A) compared to normal BRAF (1799T).
34 shows the Cpf1 target nucleotide sequence for target-specific deletion of the VEGFA gene. Specifically, A of FIG. 34 shows the target sequence in angiogenic endothelial factor (VEGFA) (blue: target 24nt sequence of CRISPR-Cpf1 gene scissors, red: PAM required for target recognition by CRISPR-Cpf1 gene scissors ( TTTN) sequence). 34B is a non-target nucleotide sequence similar to the VEGFA target nucleotide sequence, and the nucleotide sequences marked in red in the nucleotide sequence represent mismatched bases different from the Cpf1 guide nucleotide sequence.
Figure 35 shows the VEGFA gene specificity experiment compared to the off-target of Cpf1 using a chimeric DNA-RNA guide (NC: negative control, control group not treated with genetic scissors, WT: Cas12a using a guide in the form of RNA previously used , D+8: Cas12a in which 8 nt at the 3' end of the guide in the existing RNA form was substituted with DNA Asterisk: Display of truncated DNA VEGFA on-target: Within VEGFA Target sequence, VEGFA off-target: off-target sequence similar to VEGFA target sequence).
36 shows the enhancement of VEGFA gene targeting specificity compared to non-targeting of Cpf1 using a chimeric DNA-RNA guide. Figure 36A shows the cleavage comparison experiment of conventional WT-crRNA and chimeric DNA-RNA substituted with 8 DNAs for VEGFA on-target and VEGFA off-target, and B in Figure 36 shows the value calculated from A as the target Specificity (cleavage efficiency for VEGFA on-target / cleavage efficiency for VEGFA off-target) is shown (NC: negative control, control group not treated with genetic scissors, WT-crRNA: previously used RNA type guide Cas12a processing, D+8-crRNA: processing Cas12a in which 8 nt at the 3' end of the existing RNA guide was substituted with DNA VEGFA on-target: target nucleotide sequence in angiogenic endothelial factor (VEGFA) , VEGFA off-target: non-target sequence similar to VEGFA target sequence).

Hereinafter, the present invention will be described in more detail through one or more embodiments. However, these examples are intended to illustrate the present invention by way of example, 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, AsCpf1 ( Acidaminococcus sp. Cpf1) recombinant protein, whose interaction with the guide RNA was structurally well identified among known Cpf1 proteins, was first purified.

Specifically, The pET28a-Cas12a(AsCpf1) bacterial expression vector was transformed into E.coli BL21(DE3) strain, and then cultured at 37°C until OD=0.6. 48 hours after inoculation with IPTG, the bacterial cells were precipitated, the culture medium was removed, and the remaining precipitate was mixed with bufferA [20 mM Tris-HCl (pH8.0), 300 mM NaCl, 10 mM β-mercaptoethanol, 1% TritonX-100, 1 mM PMSF]. redissolved. Thereafter, the bacterial cell membrane was broken by sonication for 3 minutes on ice, and the cell lysate was harvested after centrifugation (5000 rpm, 10 min). Then, the Ni-NTA resin pre-washed with bufferB [20mM Tris-HCl (pH8.0), 300nM NaCl] was mixed with the sonicated intracellular solution, and incubated at 4°C for 1 hour and 30 minutes. while stirring. After bacterial cell precipitation, non-specific binding components were removed by washing with 10-fold volume using bufferB [20mM Tris-HCl (pH8.0), 300nM NaCl], and bufferC [20mM Tris-HCl (pH8.0), 300nM NaCl]. HCl (pH 8.0), 300 nM NaCl, 200 mM Imidazole] was used to elute the AsCpf1 protein to purify a highly pure protein with DNA cutting activity. The eluted protein was exchanged with bufferE [200mM NaCl, 50mM HEPES (pH7.5), 1mM DTT, 40% glycerol] using centricon (Amicon Ultra,), and then aliquoted and stored at -80°C.

Chimeric DNA-RNA guides were produced to order (bioneer) for batch synthesis 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 marked in bold in Tables 2 to 4 correspond to DNA nucleotide sequences.

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

target gene location division Base sequence (5'→3') sequence number 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 UGAUGUCCAUGUCUGUUACUCG 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') sequence number 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 AAUUUCUACUCUUGUAGAUGUCGGCAUGGCCCCAUTCGCACGG 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') sequence number 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 in which the interaction between the Cpf1 protein and the guide is presented, in order to reduce the binding energy of the target DNA sequence and the guide RNA, the part recognized as a proto-spacer formed by DNA-RNA junction was replaced with a DNA sequence ( Fig. 1). In addition, it was also confirmed whether the guide can be recognized as the entire DNA structure including the proto-spacer region. When four guide sequences were sequentially substituted with DNA starting from the 3' end, the cleavage efficiency of the target gene DNA ( DNMT1, CCR5 ) was compared and analyzed.

Specifically, PCR amplicons ( DNMT1, CCR5, FANCF, GRIN2B, EMX1) were obtained from genomic DNA (HEK293FT) using DNA primers (Table 5) corresponding to each locus (DNMT1, CCR5). Base sequences of forward adapter and reverse adapter primers used in next-generation sequencing (NGS) are shown in bold, respectively.

primer Primer nucleotide sequence (5'→3') sequence number hDNMT1 on-target F1 GTTGACACGTGTCAAGTGCTTA 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 AGAGAGGGGGGTCATAGACTTT 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 GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCTGCTTCGTGGCAATGCGCCAC SEQ ID NO: 200

Purified recombinant AsCpf1 and chimeric RNA-DNA guides (Bioneer) or purified crRNA corresponding to each locus synthesized in Example 1 were premixed, and in cleavage buffer (NEB3, 10 μl volume) conditions, 1 Incubated at 37°C for 1 hour. After the reaction, 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 efficiency of DNA cleavage was calculated according to the following formula by measuring the bend intensity profile of the cleaved image pattern using the imageJ program.

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

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

Example 3. Analysis of genome cleavage efficiency according to DNA substitution of Cpf1 RNA guide Seed part

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

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

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

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

Through the results of Examples 2 and 3, it was confirmed that DNA replacement in the 3' region (up to approximately +10) of the guide is effective in maintaining the cleavage activity of the target DNA compared to DNA replacement in the seed or 5' region of the guide. . Therefore, in an 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. Cleavage specificity analysis by target gene using chimeric DNA-RNA guide

In order to screen effective guides 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, by selecting a similar nucleotide sequence (off-target) for each gene target nucleotide sequence (on-target) in silico (Cas-offinder) method, on-target and off-target cleavage efficiencies of Example 2 Each method was calculated (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, about 10 substitutions, specifically 8 substitutions, without on-target cleavage compensation It was confirmed that the specificity increased up to (FIG. 12A). In the case of single base DNA substitutions in the seed region, changes up to 10 from PAM tended to decrease target DNA cleavage specificity, and in particular, consecutive changes within the seed region significantly increased on-target compensation, resulting in off-target cleavage. It was confirmed that the contrast specificity showed a sharp decrease trend (FIG. 12B).

In the case of targeting the CCR5 gene sequence, on-target cleavage was similar to that of the DNMT1 target upon sequential 3' end DNA substitution of the guide, and non-specific cleavage was not measured, confirming that the specificity showed a similar trend (FIG. 7 ). When the CCR5 gene was cleaved by applying the guide single base DNA substitution of the seed region, the specificity was reduced at the 2nd, 4th, 5th, and 7th positions from the PAM, and the rest were measured to a level similar to that of the wild-type RNA guide (FIG. 8). .

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

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

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

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

When replacing the 3' end DNA of the chimeric DNA-RNA guide, there is a possibility of degradation of the chimeric DNA-RNA guide by the 3' end DNA exonuclease.

Therefore, in order to improve the efficiency of genome editing of chimeric DNA-RNA guides with 3' end DNA substitution, the 3' end of the chimeric DNA-RNA guide is modified with phosphorothioate (PS), so that the intracellular genome It was confirmed that the calibration efficiency could be improved.

First, cleavage of the nucleotide sequence 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 a cleavage activity similar to or better than that of the wild-type RNA type guide was induced even when the guide modified with PS at the 3' end was used (FIG. 16). On the other hand, it was confirmed that non-target specific cleavage was reduced 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 nucleotide sequence within the same gene. Specifically, the HEK293FT cell line (ATCC) was subcultured in DMEM medium (DMEM (Gibco) with 10% FBS (Gibco)) every 48 hours at 37°C and 5% CO ₂ while confluency was 70%. was maintained, and an electroporation kit (amaxa, V4XC-2032) was used for chimeric cell transfection. After mixing with the Cpf1-chimeric guide pre-mixed complex using 10 ⁵ cells, electric shock (program: CM-130) was applied under conditions of electroporation buffer (Cpf1: 60 pmol, crRNA: 240 pmol). Thereafter, the transfected cells were transferred to 500 μl of a DMEM medium solution in a 24-well plate pre-incubated for 30 minutes at 37° C. in 5% CO ₂ conditions and cultured under the same conditions (37° C. and 5% CO ₂ ). The chimeric RNA-DNA guide and recombinant AsCpf1 complex were delivered into cells (HEK293FT), and 48 hours later, genomic DNA was isolated using a genomic DNA purification kit (Qiagen, DNeasy Blood & Tissue Kit).

In order to confirm whether genomic DNA is 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 machine. Annealing (reannealing) was performed (gradual decrease by 1 °C from 98 °C to 25 °C, 20 minutes). Incubation using purified recombinant T7E1 enzyme (NEB, M0302S) in a 10 μl volume at 37° C. for 25 minutes in digestion buffer (50 mM NaCl, 10 mM Tris-HCl (pH7.9), 10 mM MgCl ₂ , 1 mM DTT) did After the reaction, the reaction was stopped by mixing stop buffer (100 mM EDTA, 1.2% SDS), DNA cleavage was confirmed by 2% agarose gel electrophoresis, and the correction efficiency was measured with ImageJ program.

In addition, in order to confirm the correct nucleotide sequence of the correction site of the target gene , nested PCR (denaturation: 98 °C for 30 sec, primer annealing: 58 °C for 30 sec, elongation: 72 °C for 30 sec, 35 cycles) to insert adapter and index sequences at both ends of the amplicon (denaturation: 98 °C for 30 sec) , Primer annealing: 62°C for 15 sec, elongation 72°C for 15 sec, 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 the Cas-Analyzer code and the calibration efficiency (%) was calculated.

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

Example 6. Comparison of DNMT1 target sequence indel induction efficiency in genes of AsCpf1, LbCpf1 and FnCpf1 based on chimeric DNA-RNA guide using plasmid

In addition to AsCpf1, it was confirmed whether the chimeric DNA-RNA guide could be applied to Cpf1 derived from other microbial species.

Specifically, LbCpf1 and FnCpf1 were purified into proteins by expressing self-produced expression plasmids in bacteria in the same manner as in AsCpf1 by the method of Example 2. In addition, the plasmid containing the target DNMT1 sequence was prepared by mimicking the nucleotide sequence on genomic DNA as it is, and delivered intracellularly by electroporation like the purified Cpf1 gene editing protein. The correction of was analyzed by the next-generation sequencing method of Example 2.

As a result, similar cleavage patterns were observed for the target and non-target sequences (FIG. 20), and it was confirmed that the target specificity was also improved (FIG. 21).

Example 7. Confirmation of improved intracellular genome editing efficiency of chimeric DNA-RNA guide-based Cpf1 gene scissors by using SpCas9 nickase (D10A) mixture

In order to confirm whether the DNA double helix in the actual genome in cells is due to the negative supercoil form wound around histone proteins, 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 the negative supercoil target sequence. Using this together with a chimeric DNA-RNA guide with unmodified 3' ends and a chimeric DNA-RNA guide with PS-modified 3' ends, the intracellular genome editing efficiency was measured in the same manner as in Example 5. (FIG. 22).

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

DNMT1, GRIN2B, and HPRT1 of AsCpf1 using chimeric DNA-RNA guides (contiguous DNA substitutions of 4nt and 8nt lengths at the 3' end of crRNA, +4 and +8 are the number of substituted DNAs) in the same way as in the previous salpin method , The target specificity of the RPL32P3 gene nucleotide sequence was confirmed, and the results are shown in FIG. 23 .

23A to D show the results of confirming the target specificity of the DNMT1, GRIN2B, HPRT1, and RPL32P3 gene sequences, respectively (On/Off1 ratio (blue).

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

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

In order to confirm the possibility of acting in various forms of the system using the chimeric DNA-RNA guide according to the present invention, as shown in FIG. Upon delivery, an experiment was performed to compare target/off-target sequence editing efficiencies on the plasmid.

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

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

Example 10. Cas12a (Cpf1) gene editing activity by combined use of CCR5 gene target 3' terminal PS (phosphorothioate) improved chimeric guide and nickase SpCas9

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

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

The experimental results are shown in B to C of FIG. 25 . 25B shows the results of analyzing the efficiency of NGS genome editing by co-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 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 by combining the target 3' terminal phosphorothioate (PS) improved chimeric guide with the nickase SpCas9, including the 8 DNA 3' terminal sequences according to the present invention. confirmed to appear.

Example 11. Application of gene therapy for targeted treatment of viral (eg, HIV) infectious diseases

Because HIV is a retrovirus, it is highly susceptible to modification of this protein, so it cannot be targeted properly, so there is no perfect cure yet. Accordingly, CRISPR-Cas12a (Cpf1) genetic scissors were used to selectively remove the CCR5 gene in T cells, and it was confirmed whether effective gene therapy in the form of ex-vivo injection by human subject autologous T cell correction could be used in the future. .

Specifically, pET28a-Cas12a (AsCpf1) bacterial expression vector was transfected into E.coli BL21 (DE3) species for purification of recombinant protein AsCpf1 (Acidaminococcus sp. After conversion, it was incubated at 37˚C until OD=0.6. 48 hours after IPTG inoculation, bacterial cells were precipitated, the culture medium was removed, and the remaining precipitate was reconstituted with bufferA [20 mM Tris-HCl (pH8.0), 300 mM NaCl, 10 mM β-mercaptoethanol, 1% TritonX-100, 1 mM PMSF]. dissolved. Thereafter, the bacterial cell membrane was broken through sonication (ice, 3 min), and cell lysate was harvested after centrifugation (5000 rpm, 10 min). First, Ni-NTA resin pre-washed with buffer B [20mM Tris-HCl (pH8.0), 300nM NaCl] and sonicated intracellular solution were mixed and stirred for 1 hour and 30 minutes in a cold room (4C). After bacterial cell precipitation, non-specific binding components were removed by washing with 10-fold volume using bufferB [20mM Tris-HCl (pH8.0), 300nM NaCl], and 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 centricon (Amicon Ultra,), and then aliquoted and stored at -80˚C. As a result, high-purity proteins having DNA cleavage activity were purified, and the results are shown in FIG. 26 .

As can be seen in Figure 26, the result of purification of the gene editing CRISPR-Cas12a (Cpf1) recombinant protein using Affinity column (Ni-NTA resin). AsCpf1 ( Acidaminococcus sp. Cpf1) and LbCpf1 (Cpf1), each of which has a (6X)His-tag attached to the N-terminus, were isolated/purified in bacterial cells to obtain activated gene clipping proteins with a purity of 90% or more.

The chimeric DNA-RNA guide necessary for the CCR5 gene targeting experiment was custom-made (bioneer) according to the target nucleotide sequence in the target CCR5 gene, and the corresponding sequence is shown in Table 6 and FIG. 27 as follows.

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

In addition, as can be seen in A of FIG. 27, the Cpf1 target nucleotide sequence for target-specific removal of the CCR5 gene is shown, and the red color represents the PAM (TTTN) nucleotide sequence, and the yellow color represents the target nucleotide sequence. In B, the CCR5 target sequence (on-target) and similar off-target sequence (off-target) information are shown, and the part that mismatches with the guide RNA in the off-target sequence is marked in red. .

Based on the above information, a CCR5 gene targeting experiment was performed in animal cells of Cpf1 using a chimeric DNA-RNA guide. For in vivo application of Cpf1 using a specific chimeric DNA-RNA guide with high target DNA cleavage specificity, it was confirmed 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 medium in which 10% FBS (Gibco) was added to DMEM (Gibco) was used, and 70% confluency of the culture plate was maintained through subculture every 48 hours in an environment of 37°C and 5% CO2. Electroporation (Lonza, V4XC-2032) was used for efficient delivery of the gene scissors vector into the nucleus, and As/LbCp1 (500ng), nCas9 (D10A (100ng)), crRNA (150 pmol), sgRNA (15 pmol) vector and guide RNA were delivered. 12 hours after electroporation, the same amount of crRNA and sgRNA was delivered into cells using 2.8 ul of Lipofectamine 3000 (ThermoFisher) and 2.0 μl of P3000 reagent, and delivered again with the same method and time interval, for a total of 2 additional guides. RNA delivery was performed. After electroporation, 72 hours later, genomic DNA of the HEK293FT cell line was extracted. Using the extracted genomic DNA, target genes and expected off-target gene locations were analyzed through targeted amplicon sequencing. The saved Fastq file was analyzed with the Cas-Analyzer code and the calibration efficiency (%) was calculated.

The experimental results are shown in FIGS. 28 and 29. The above results show the CCR5 gene targeting specificity in animal cells of Cpf1 gene scissors by using chimeric DNA-RNA guides.

Specifically, as a result of targeting a specific nucleotide sequence (table) in the CCR5 gene in a human cell line (HEK293FT) using Cpf1 genetic scissors, nickase was simultaneously used when using +8DNA crRNA, in which the 3' end of guide RNA was substituted with DNA rather than WT crRNA The efficiency was maximized by treatment (Figs. 28 A and 29 A). On the other hand, the rate of indel formation by inducing off-target cleavage 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 genetic scissors, targeting specificity was 2 when using +8DNA crRNA in which the 3' end of guide RNA was substituted with DNA than WT crRNA. It was confirmed that it increased more than twice (FIG. 28 C).

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

Example 12. Application of gene therapy for oncogenic gene target therapy

Genes with an underlying cause that can cause progression to cancer in the human body are known as oncogenes. These proto-oncogenes are mutated by active oxygen or radiation, and when their properties are converted into oncogenes, they cause endless cell division without being controlled by the control system. They multiply and form cancer. Accordingly, by using CRISPR-Cpf1 gene scissors with improved target specificity due to reduced non-targeting, it is possible to target oncogenes that cause cancer in humans and selectively remove them compared to proto-oncogenes required in the human body. An experiment was conducted to confirm that

Specifically, as shown in FIG. 30, Cpf1 gene scissors with improved target specificity that can cut only the oncogenic gene BRAF (1799T>A) without cutting the normal gene BRAF are applied to kill cancer cells by removing the oncogene that is the source of cancer cells Checked if it could be done.

The chimeric DNA-RNA guide required for the BRAF mutant gene (1799T>A) target experiment was custom-made (bioneer) for batch synthesis according to the nucleotide sequence targeting the point mutation (1799T>A) in the target BRAF gene, which is shown in Table 7 and 31.

In Table 7 above, the PAM nucleotide sequence (TTTN) in the target DNA gene is underlined, and the DNA nucleotide sequence of the chimeric guide is 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 A of FIG. 31, orange: 24nt nucleotide sequence target of CRISPR-Cpf1 gene scissors, blue: PAM (TTTN) sequence required for target recognition by CRISPR-Cpf1 gene scissors, red: normal gene (1799T ), the occurrence of mutation into an oncogenic gene (1799T>A) was specifically described. In addition, as can be seen in B of FIG. 31, point mutations occurred within the BRAF gene. MT: mutant gene (oncogene), WT: normal gene (proto-oncogene) were described.

In-vitro level oncogene specific cleavage experiments were 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. Each base sequence of the normal BRAF gene (1799T) and the oncogene BRAF (1799T>A) was synthesized and inserted into a T-vector, and PCR was performed on each containing the target sequence to obtain an amplicon. Afterwards, hBRAF_Mutation_amplicon 2μg, hBRAF_wild-type_amplicon 2μg, AsCpf1 or LbCpf1 protein 2.8μg, crRNA (WT) 600ng or chimeric DNA-RNA (D+8) 600ng were added to cleavage buffer (NEB3.1 buffer, deionized water up to 10μl) After 1 hour incubation in a 37°C incubator, the band pattern was confirmed by electrophoresis at 200V-20min in an agarose 2% gel. Then, 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), as shown in FIG. 32, wild-type and chimeric DNA for the oncogene BRAF (1799T>A) and the normal BRAF gene (1799T) The cleavage efficiency of Cpf1 using each -RNA guide (D+8) was compared and analyzed.

For the oncogene BRAF (1799T>A), the DNA cleavage efficiency of Cpf1 gene scissors using wild-type and chimeric DNA-RNA guides (D+8) was similar, and for the normal BRAF gene (1799T), When the meric DNA-RNA guide (D + 8) was used, DNA cleavage efficiency was significantly reduced (Fig. 33 A), and thus Cpf1 gene scissors using the chimeric DNA-RNA guide (D + 8) compared to the normal BRAF gene (1799T). The oncogene BRAF(1799T>A) showed an increase in specificity (Fig. 33 B).

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 CRISPR-Cpf1 genetic scissors with improved target specificity, the vascular endothelial factor (VEGFA) gene is directly targeted and fundamentally removed at the DNA level confirmed that it could be done.

Chimeric DNA-RNA guides necessary for the VEGFA gene targeting experiment were manufactured according to the target nucleotide sequence in the target VEGFA gene, and were produced to order, which are shown in Table 8 and FIG. 34.

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

In addition, FIG. 34 shows the Cpf1 target nucleotide sequence for target-specific deletion of the VEGFA gene. 34A shows the target nucleotide sequence in angiogenic endothelial factor (VEGFA), blue: target 24nt nucleotide sequence of CRISPR-Cpf1 genetic scissors, red: PAM (TTTN) nucleotide sequence required for target recognition by CRISPR-Cpf1 genetic scissors indicates 34B shows off-target nucleotide sequences similar to the VEGFA target nucleotide sequence, and nucleotide sequences marked in red in the nucleotide sequence represent mismatched bases different from the Cpf1 guide sequence.

In-vitro level VEGFA gene specific cleavage experiments were performed.

In- An in vitro cleavage assay was performed. Each of the hVEGFA target gene (on-target) and non-target gene (off-target) nucleotide sequences were synthesized and inserted into a T-vector, and PCR was performed on each of the target nucleotide sequences to obtain an amplicon. Then, 600ng of hVEGFA_on-target amplicon, 600ng of hVEGFA_off-target amplicon, 2.8μg of AsCpf1-FLAG protein, 600ng of crRNA(WT), and 600ng of crRNA(D+8) were mixed with cleavage buffer (NEB3.1 buffer, DeIonized Water up to 10μl) After 1 hour incubation in a 37 ℃ incubator by mixing, the band pattern was confirmed by electrophoresis in agarose 2% gel at 200V-20min. In addition, DNA cleavage efficiency (cleavage efficiency = cleaved band intensity / total intensity X100) was calculated with ImageJ software.

In order to confirm off-target specificity for the target VEGFA gene, wild-type and chimeric DNA-RNA guides (D +8) were 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 the on-target sequence in the target gene was similar, and the off-target sequence (off-target) target), DNA cleavage efficiency was significantly reduced when the chimeric DNA-RNA guide (D + 8) was used (Fig. 36 A), so that Cpf1 gene scissors using the chimeric DNA-RNA guide (D + 8) It was shown that the specificity of the nucleotide sequence (on-target) within the target gene was increased compared to the off-target sequence (FIG. 36 B).

So far, the present invention has been examined focusing on the embodiments. Those skilled in the art to which the present invention pertains will be able to 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 should be considered from an illustrative rather than a limiting point of view. The scope of the present invention is shown in the claims rather than the foregoing description, and all differences within the equivalent range should be construed as being included in the present invention.

<110> Korea Research Institute of Bioscience and Biotechnology <120> Composition for Genome Editing or Inhibiting Gene Expression comprising Cpf1 and Chimeric DNA-RNA Guide <130> PN190345 <150> KR 10-2019-0170035 <151> 2019-12-18 <160> 212 <170> KoPatentIn 3.0 <210> 1 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA1_AsCpf1_target DNA <400> 1 tttcctgatg gtccatgtct gttactcg 28 <210> 2 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA1_AsCpf1_guide <400> 2 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_ crRNA2_AsCpf1_target DNA <400> 3 tttcctgatg gtccatgtct gttactcg 28 <210> 4 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 4 aauuucuacu cuuguagauc ugauggucca ugucuguuac TCG 43 <210> 5 <211> 28 <212> DNA <213> Artificial sequence <220> <223> hdnmt1_crila3_ascpf1_target DNA <400> 211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 6 aauuucuacu cuuguagauc ugauggucca ugucugttac tcg 43 <210> 7 <211> 28 <212> DNA <213> Artificial Sequence <220> <223 > hDNMT1_crRNA3-2_AsCpf1_target DNA <400> 7 tttcctgatg gtccatgtct gttactcg 28 <210> 8 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400 > 8 aauuucuacu cuuguagauc ugauggucca ugucugttac tcg 43 <210> 9 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-3_AsCpf1_target DNA <400> 9 tttcctgatg gtccatgtct gttactcg 28 <210> 10 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-3_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 10 aauuucuacu cuuguagauc ugauggucca ugucugttac tcg 43 <210> 11 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-4_AsCpf1_target DNA <400> 11 tttcctgatg gtccatgtct gttactcg 28 <210> 12 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-4 _AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 12 aauuucuacu cuuguagauc ugauggucca ugucugttac tcg 43 <210> 13 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA4_AsCpf1_target DNA <400> 13 tttcc tgatg gtccatgtct gttactcg 28 <210> 14 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA4_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 14 aauuucuacu cuuguagauc ugauggucca ugtctgttac tcg 43 <210> 15 <211 > 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA5_AsCpf1_target DNA <400> 15 tttcctgatg gtccatgtct gttactcg 28 <210> 16 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA5_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 16 aauuucuacu cuuguagauc ugauggucca tgtctgttac tcg 43 <210> 17 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA6_AsC pf1_target DNA <400> 17 tttcctgatg gtccatgtct gttactcg 28 <210> 18 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA6_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 18 aauuucuacu cuuguagauc ugatggtcca tgtctgttac tcg 43 <210> 19 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA7_AsCpf1_target DNA <400> 19 tttcctgatg gtccatgtct gttactcg 28 <210> 20 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> < 223> hDNMT1_crRNA7_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 20 aauuucuacu cuuguagauc tgatggtcca tgtctgttac tcg 43 <210> 21 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 _crRNA8_AsCpf1_target DNA <400> 21 tttcctgatg gtccatgtct gttactcg 28 <210> 22 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA8_AsCpf1_guide <400> 22 aatttctact cttgtagatc tgatggtcca tgtctgttac tcg 43 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA9_AsCpf1_target DNA <400> 23 tttcctgatg gtccatgtct gttactcg 28 <210> 24 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA9_AsC pf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 24 aattucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 25 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA10_AsCpf1_target DNA <400> 25 tttcctgatg gt ccatgtct gttactcg 28 < 210 > 26 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA10_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 26 aatttctacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 27 <211> 28 < 212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA11_AsCpf1_target DNA <400> 27 tttcctgatg gtccatgtct gttactcg 28 <210> 28 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_ crRNA11_AsCpf1_chimeric guide < 220> <223> Synthetic oligonucleotide <400> 28 aatttctact ctuguagauc ugauggucca ugucuguuac ucg 43 <210> 29 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA12_AsCpf1_target DNA <400> 29 tttcct gatg gtccatgtct gttactcg 28 <210> 30 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA12_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 30 aatttctact cttgtagauc ugauggucca ugucuguuac ucg 43 <210> 31 <2 11> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA13_AsCpf1_target DNA <400> 31 tttcctgatg gtccatgtct gttactcg 28 <210> 32 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDN MT1_crRNA13_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 32 aatttctact cttgtagatc ugauggucca ugucuguuac ucg 43 <210> 33 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA14_AsCpf1_target DNA <400> 33 tt tcctgatg gtccatgtct gttactcg 28 <210> 34 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA14_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 34 aauuucuacu cuuguagauc tgauggucca ugucuguuac ucg 43 < 210> 35 < 211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA15_AsCpf1_target DNA <400> 35 tttcctgatg gtccatgtct gttactcg 28 <210> 36 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <2 23 > hDNMT1_crRNA15_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 36 aauuucuacu cuuguagauc tgatggtcca ugucuguuac ucg 43 <210> 37 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA16 _AsCpf1_target DNA <400> 37 tttcctgatg gtccatgtct gttactcg 28 <210> 38 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA16_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 38 aauuucuacu cuuguagauc tgatgg tcca tgucuguuac ucg 43 <210> 39 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA17_AsCpf1_target DNA <400> 39 tttcctgatg gtccatgtct gttactcg 28 <210> 40 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220 > <223> hDNMT1_crRNA17_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 40 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 41 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA 18_AsCpf1_target DNA <400 > 41 tttcctgatg gtccatgtct gttactcg 28 <210> 42 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA18_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 42 aauuucuacu cuuguagauc tgauggucca ugucuguuac ucg 43 < 210> 43 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA19_AsCpf1_target DNA <400> 43 tttcctgatg gtccatgtct gttactcg 28 <210> 44 <211> 43 <212> DNA_RNA <213> Artificial Sequence ence < 220> <223> hDNMT1_crRNA19_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 44 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 45 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDN MT1_crRNA20_AsCpf1_target DNA cu uguagauc ugauggucca ugucuguuac ucg 43 <210> 47 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA21_AsCpf1_target DNA <400> 47 tttcctgatg gtccatgtct gttactcg 28 <210> 48 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA21_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 48 aauuucuacu cuuguagauc ugatggucca ugucuguuac ucg 43 <210> 49 <211> 28 <212> DNA <213> Artificial Sequence <220> <223 > hDNMT1_crRNA22_AsCpf1_target DNA <400> 49 tttcctgatg gtccatgtct gttactcg 28 <210> 50 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA22_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 50 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 51 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA23_AsCpf1_target DNA <400> 51 tttcctgatg gtccatgtct gttactcg 28 <210> 52 <211> 43 <212 >DNA_RNA <213 > Artificial Sequence <220> <223> hDNMT1_crRNA23_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 52 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 53 <211> 28 <212> DNA <213> Artificial Sequence <220> < 223> hDNMT1_crRNA24_AsCpf1_target DNA <400> 53 tttcctgatg gtccatgtct gttactcg 28 <210> 54 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA24_AsCpf1_chimeric guide <220> < 223> Synthetic oligonucleotide <400> 54 auuucuacu cuuguagauc ugauggtcca ugucuguuac ucg 43 <210> 55 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA25_AsCpf1_target DNA <400> 55 tttcctgatg gtccatgtct gttactcg 28 <210> 56 < 211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA25_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 56 aauuucuacu cuuguagauc ugauggucca gucuguuac ucg 43 <210> 57 <211> 28 <212> DNA <213> Artificial Sequence <22 0 > <223> hDNMT1_crRNA26_AsCpf1_target DNA <400> 57 tttcctgatg gtccatgtct gttactcg 28 <210> 58 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA26_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 58 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 59 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA27_AsCpf1_target DNA <400> 59 tttcctgatg gtccatgtct gttactcg 28 <210> 60 <211> 43 <212 > DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA27_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 60 aauuucuacu cuuguagauc ugauggucca ugucuguuac ucg 43 <210> 61 <211> 28 <212> DNA <213> Artificial Sequence ence <220> <223> hCCR5_crRNA51_AsCpf1_target DNA <400> 61 tttgtgcaca gggtggaaca agatggat 28 <210> 62 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA51_AsCpf1_guide <400> 62 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 63 <211> 28 <212> DNA <213> Artificial sequence <220> <223> hcCr5_crrNA52_ASCPF1_TARGET DNA <400> 1> 43 <212> DNA_RNA <213> Artificial sequence <220> <223> hCCR5_crRNA52_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 64 aauuucuacu cuuguagauu gcacagggug gaacaagaug gat 43 <210> 65 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR 5_crRNA53_AsCpf1_target cu uguagauu gcacagggug gaacaagatg gat 43 <210> 67 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA54_AsCpf1_target DNA <400> 67 tttgtgcaca gggtggaaca agatggat 28 <210> 68 <211> 43 <212> DNA_RNA <213 > Artificial Sequence <220> <223> hCCR5_crRNA54_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 68 aauuucuacu cuuguagauu gcacagggug gaacaagatg gat 43 <210> 69 <211> 28 <212> DNA <213> Artificial Sequence <220> <22 3 > hCCR5_crRNA55_AsCpf1_target DNA <400> 69 tttgtgcaca gggtggaaca agatggat 28 <210> 70 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA55_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 70 aauuucuacu cuuguagauu gcacagggtg gaacaagatg gat 43 <210> 71 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA56_AsCpf1_target DNA <400> 71 tttgtgcaca gggtggaaca agatggat 28 <210> 72 <211> 43 <212> DNA_RNA < 213> Artificial Sequence <220> <223> hCCR5_crRNA56_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 72 aauuucuacu cuuguagauu gcacagggtg gaacaagatg gat 43 <210> 73 <211> 28 <212> DNA <213> Artificial Sequence <22 0> <223> hCCR5_crRNA57_AsCpf1_target DNA <400> 73 tttgtgcaca gggtggaaca agatggat 28 <210> 74 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA57_AsCpf1_chimeric guide <220> <2 23> Synthetic oligonucleotide <400> 74 aauuucuacu cuuguagaut gcacagggtg gaacaagatg gat 43 <210> 75 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA58_AsCpf1_target DNA <400> 75 tttgtgcaca gggtggaaca agatggat 28 <210> 76 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA58_AsCpf1_guide <400> 76 aatttctact cttgtagatt gcacagggtg gaacaagatg gat 43 <210> 77 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA6 1_AsCpf1_target DNA <400 > 77 tttgtgcaca gggtggaaca agatggat 28 <210> 78 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA61_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 78 aauuucuacu cuuguagaut gca cagggug gaacaagaug gau 43 < 210> 79 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA62_AsCpf1_target DNA <400> 79 tttgtgcaca gggtggaaca agatggat 28 <210> 80 <211> 43 <212> DNA_RNA <213> Artificial Sequence < 220> <223> hCCR5_crRNA62_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 80 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 81 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR 5_crRNA63_AsCpf1_target DNA <400> 81 tttgtgcaca gggtggaaca agatggat 28 <210> 82 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA63_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 82 aauuucuacu cuugu agauu gcacagggug gaacaagaug gau 43 <210> 83 <211> 28 <212> DNA <213> Artificial sequence <220> <223> hcCr5_CRRNA64_ASCPF1_TARGET DNA <400> 83 211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hcCr5_crila64_ascpf1_chimeric guide <220> <223> Synthetic oligonucleotide GAUG GAU 43 <210> 85 <211> 28 <212> DNA <213> Artificial sequence <220> <223> hCCR5_crRNA65_AsCpf1_target DNA <400> 85 tttgtgcaca gggtggaaca agatggat 28 <210> 86 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA65_AsCpf1_chimeric guide <220> <223> Synthetic oligo nucleotide <400> 86 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 87 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA66_AsCpf1_target DNA <400> 87 tttgtgcaca gggtggaaca agatggat 28 <210> 88 <211> 43 <212> DNA_ RNA <213 > Artificial Sequence <220> <223> hCCR5_crRNA66_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 88 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 89 <211> 28 <212> DNA <213> Artificial Sequence <220> < 223> hCCR5_crRNA67_AsCpf1_target DNA <400> 89 tttgtgcaca gggtggaaca agatggat 28 <210> 90 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA67_AsCpf1_chimeric guide <220> <2 23> Synthetic oligonucleotide <400> 90 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 91 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA68_AsCpf1_target DNA <400> 91 tttgtgcaca gggtggaaca agatggat 28 <210> 92 <21 1> 43 <212> DNA_RNA <213> Artificial sequence <220> <223> hcCr5_crida68_ascpf1_chimeric guide <220> <223> Synthetic oligonucleotide Caggggug Gaacagaug Gau 43 <210> 93 <211> 28 <212> DNA <213> Artificial sequence <220 > <223> hCCR5_crRNA69_AsCpf1_target DNA <400> 93 tttgtgcaca gggtggaaca agatggat 28 <210> 94 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA69_AsCpf1_chimeric guide <220> < 223> synthetic oligonucleotides <400> 94 aauuucuacu cuuguagauu gcacagggug gaacaagaug gau 43 <210> 95 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_crRNA70_AsCpf1_target DNA <400> 95 tttgtgcaca gggtggaaca agatggat 28 <210> 96 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5_crRNA70_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 96 aauuucuacu cuuguagauu gcacagggtg gaacaagaug gau 43 <210> 97 <211> 28 <212> DNA <2 13 > Artificial Sequence <220> <223> hFANCF_crRNA81_AsCpf1_target DNA <400> 97 tttggtcggc atggccccat tcgcacgg 28 <210> 98 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hFANCF_crRNA81_AsCpf1_ chimeric guide <400> 98 aauuucuacu cuuguagaug ucggcauggc cccauucgca cgg 43 <210> 99 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hFANCF_crRNA82_AsCpf1_target DNA <400> 99 tttggtcggc atggccccat tcgcacgg 28 <210> 100 <211> 43 <212> DNA_RNA < 213> Artificial Sequence <220> <223> hFANCF_crRNA82_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 100 aauuucuacu cuuguagaug ucggcauggc cccauucgca cgg 43 <210> 101 <211> 28 <212> DNA <213> Artificial Sequence <2 20> <223> hFANCF_crRNA82-2_AsCpf1_target DNA <400> 101 tttggtcggc atggccccat tcgcacgg 28 <210> 102 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hFANCF_crRNA82-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 102 aauuucuacu cuuguagaug ucggcauggc cccautcgca cgg 43 <210> 103 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hFANCF_crRNA83_AsCpf1_target DNA <400> 103 tttggtgctc aatgaaagga gataaggt 28 <210> 104 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hFANCF_crRNA83_AsCpf1_guide <400> 104 aauuucuacu cuuguagaug ugcucaauga aaggagauaa ggu 43 <210> 105 <211> 28 <212> DNA <213> Artificial Sequence <220> <22 3> hFANCF_crRNA84_AsCpf1_target DNA <400> 105 tttggtgctc aatgaaagga gataaggt 28 <210> 106 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hFANCF_crRNA84_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 106 aauuucuacu cuuguagaug ugcucaauga aaggagauaa ggt 43 <210> 107 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hFANCF_crRNA84-2_AsCpf1_target DNA <400> 107 tttggtgctc aatgaaagga gataaggt 28 <210> 108 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hFANCF_crRNA84-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 108 aauuucuacu cuuguagaug ugcucaauga aaggagataa ggt 43 <210> 109 <211> 28 <212> DNA <213> Artificial Sequence ence <220> <223> hEMX1_crRNA85_AsCpf1_target DNA <400> 109 tttgtcctcc ggttctggaa ccacacct 28 <210> 110 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hEMX1_crRNA85_AsCpf1_guide <4 00> 110 aauuucuacu cuuguagauu ccuccgguuc uggaaccaca ccu 43 <210> 111 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hEMX1_crRNA86_AsCpf1_target DNA <400> 111 tttgtcctcc ggttctggaa ccacacct 28 <210> 112 <211> 43 <212> DNA_RNA <213 > Artificial Sequences <220> <223> hEMX1_crRNA86_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 112 aauuucuacu cuuguagauu ccuccgguuc uggaaccaca cct 43 <210> 113 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hEMX1_crRNA86 -2_AsCpf1_target DNA <400> 113 tttgtcctcc ggttctggaa ccacacct 28 <210> 114 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hEMX1_crRNA86-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 114 aauuucuacu cuuguagauu ccuccgguuc uggaaccaca cct 43 <210> 115 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2-2_AsCpf1_target DNA <400> 115 tttcctgatg gtccatgtct gttactcg 28 <210> 116 <211> 43 < 212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 116 aauuucuacu cuuguagauc ugauggucca ugucuguuac tcg 43 <210> 117 <211> 28 <212> DNA <213 > Artificial Sequence <220> <223> hDNMT1_crRNA2-3_AsCpf1_target DNA <400> 117 tttcctgatg gtccatgtct gttactcg 28 <210> 118 <211> 47 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2-3_As Cpf1_chimeric guide <220 > <223> Synthetic oligonucleotide <400> 118 aauuucuacu cuuguagauc ugauggucca ugucuguuac tcgtttt 47 <210> 119 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2-4_AsCpf1_target DNA <400> 119 tttcctgatg gtccatgtct gttactcg 28 <210> 120 <211> 46 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA2-4_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 120 aauuucuacu cuuguagauc ugauggucca ugucuguuac tcgttt 46 < 210 > 121 <211> 28 <212> DNA <213> Artificial sequence <220> <223> hdnmt1_cruna3-2_ASCPF1_TARGET DNA <400> 121 ttttccatgtct GTTACTCG 28 <210> 122 <211> 43 <212> DNA_RNA <213> Artificial sequence <220 > <223> hDNMT1_crRNA3-2_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 122 aauuucuacu cuuguagauc ugauggucca ugucugttac tcg 43 <210> 123 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDN MT1_crRNA3 -3_AsCpf1_target DNA <400> 123 tttcctgatg gtccatgtct gttactcg 28 <210> 124 <211> 47 <212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-3_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <4 00> 124 aauuucuacu cuuguagauc ugauggucca ugucugttac tcgtttt 47 <210> 125 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-4_AsCpf1_target DNA <400> 125 tttcctgatg gtccatgtct gttactc g 28 <210> 126 <211> 46 < 212> DNA_RNA <213> Artificial Sequence <220> <223> hDNMT1_crRNA3-4_AsCpf1_chimeric guide <220> <223> Synthetic oligonucleotide <400> 126 aauuucuacu cuuguagauc ugauggucca ugucugttac tcgttt 46 <210> 127 <211> 21 <212 > DNA <213 > Artificial Sequence <220> <223> hDNMT1 on-target F1 <400> 127 gttgcacgtg tcaagtgctt a 21 <210> 128 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 on-target R1 <400> 128 ttaaaatcca gaatgcacaa agtact 26 <210> 129 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 on-target F2 <400> 129 gtgaatttgg ctcagcaggc a 21 <210> 130 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 on-target R2 <400> 130 aagcgaacct cacacaacag c 21 <210> 131 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target1 F1 <400> 131 ttgacggcag tattacaggt ag 22 <210> 132 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target1 R1 <400> 132 aaggtcaaat gccgtttaac ca 22 <210> 133 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target1 F2 <400> 133 gtagtcaggc atgagtggca 20 <210> 134 <211> 20 <212> DNA < 213> Artificial Sequence <220> <223> hDNMT1 off-target1 R2 <400> 134 tgcctctttc ccaggattct 20 <210> 135 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target2 F1 <400> 135 tctcaggcaa gtcacaactc t 21 <210> 136 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target2 R1 <400> 136 taggcacatg aaggtcaaat gc 22 <210> 137 <211 > 22 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target2 F2 <400> 137 gttacaggta gttaagcagg ca 22 <210> 138 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target2 R2 <400> 138 aaatgccata tttaaccgtg atcct 25 <210> 139 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target3 F1 <400> 139 tcatgccttt ctgggtctca t 21 <210> 140 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target3 R1 <400> 140 cacctgacct ctgtcacttt a 21 <210> 141 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off-target3 F2 <400> 141 ctgttcaggg aatggaaagt ga 22 <210> 142 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1 off- target3 R2 <400> 142 ctacatctac gctctcccca 20 <210> 143 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 on-target F1 <400> 143 aaggctgagc tgcaccatgc 20 <210> 144 <211 > 21 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 on-target R1 <400> 144 aggatgatga agaagattcc a 21 <210> 145 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 on-target F2 <400> 145 cattcactcc atggtgctat a 21 <210> 146 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 on-target R2 <400> 146 atagagccct gtcaagagtt ga 22 <210> 147 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target1 F1 <400> 147 agaaatagga gtcttcatgc cc 22 <210> 148 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target1 R1 <400> 148 tggggtcaat gagaggagta tt 22 <210> 149 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off- target1 F2 <400> 149 tagcaaggaa cctcaaagtg c 21 <210> 150 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target1 R2 <400> 150 ctgcttccaa tacaatccac ac 22 <210> 151 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target2 F1 <400> 151 tagtgctgaa agctcagaga g 21 <210> 152 <211> 20 <212> DNA <213> Artificial Sequence < 220> <223> hCCR5 off-target2 R1 <400> 152 ttcgagagcc acacatgaag 20 <210> 153 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target2 F2 <400> 153 agagagggggg tcatagactt t 21 <210> 154 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 off-target2 R2 <400> 154 accttccagc cggtatatta at 22 <210> 155 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF on-target F1 <400> 155 tgaaagcgga agtagggcct 20 <210> 156 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF on- target R1 <400> 156 ctccgcctgg gtcttcatca 20 <210> 157 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF on-target F2 <400> 157 ggaagtaggg ccttcgcgca 20 <210> 158 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF on-target R2 <400> 158 cctcctggag atttgggttc 20 <210> 159 <211> 21 <212> DNA <213> Artificial Sequence <220> < 223> hFANCF off-target1 F1 <400> 159 cctgtaagcc ctaagcaagt c 21 <210> 160 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> hFANCF off-target1 R1 <400> 160 gtcaagttct actcaaggcc at 22 <210> 161 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hFANCF off-target1 F2 <400> 161 ctgtcctgta gagacttctg g 21 <210> 162 <211> 22 <212> DNA < 213> Artificial Sequence <220> <223> hFANCF off-target1 R2 <400> 162 gtggtggagg gaagcttatt tt 22 <210> 163 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF off-target2 F1 <400> 163 tggggtaggt cttcaggaaa 20 <210> 164 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hFANCF off-target2 R1 <400> 164 cgttctaaat tctctacagt caac 24 <210> 165 <211 > 20 <212> DNA <213> Artificial Sequence <220> <223> hFANCF off-target2 F2 <400> 165 tcgtgctaag ttaccgagtt 20 <210> 166 <211> 21 <212> DNA <213> Artificial Sequence <220> < 223> hFANCF off-target2 R2 <400> 166 gcaatagttt tgagccagtg a 21 <210> 167 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B on-target F1 <400> 167 acctctgctg agcacgtttt 20 <210> 168 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B on-target R1 <400> 168 gacagcaatg ccaatgctgg 20 <210> 169 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B on-target F2 <400> 169 ctcactttgt ctggccttgc 20 <210> 170 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B on-target R2 <400 > 170 CTTCTGAGAA CGAGCTCTGC 20 <210> 171 <211> 23 <212> DNA <213> Artificial sequence <220> <223> HGRIN2B Off-Target1 <400> 171 TGACTGCAAT TTTGGTCC AT C 23 <210> 172 <211> 23 < 212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target1 R1 <400> 172 gccactgcca tttattatgt aac 23 <210> 173 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target1 F2 <400> 173 gtctaattac acttgccata cct 23 <210> 174 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target1 R2 <400> 174 gaaatccagc tgtgactaaa tatg 24 < 210> 175 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target2 F1 <400> 175 ggagcacaat gagtgttttt 20 <210> 176 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target2 R1 <400> 176 gtcttttcct ggtcacatgg 20 <210> 177 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target2 F2 <400> 177 gccagaaatg ttattccata gtag 24 <210> 178 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B off-target2 R2 <400> 178 cagtgttctt acatatcaga cac 23 <210> 179 <211> 24 < 212> DNA <213> Artificial Sequence <220> <223> hEMX1 on-target F1 <400> 179 actactcaca tccactctgt gaag 24 <210> 180 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 on-target R1 <400> 180 gagtggccag agtccagctt 20 <210> 181 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 on-target F2 <400> 181 tagaggagct aggatgcaca gca 23 <210 > 182 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 on-target R2 <400> 182 gcagcaagca gcactctgcc 20 <210> 183 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target1 F1 <400> 183 cagagcctgg agaattgata g 21 <210> 184 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target1 R1 <400> 184 ctggggagca gaataaatta ttg 23 <210> 185 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target1 F2 <400> 185 gaatagacct gggatgtgca g 21 <210> 186 <211> 21 < 212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target1 R2 <400> 186 ggaacctgaa gaatgggatt g 21 <210> 187 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target2 F1 <400> 187 agattgagat catccaccct 20 <210> 188 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target2 R1 <400> 188 aaaacagaga ggttgagtct c 21 <210 > 189 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target2 F2 <400> 189 tgtccaccat gtcccagaag 20 <210> 190 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> hEMX1 off-target2 R2 <400> 190 acaaagaagg gcatctccag 20 <210> 191 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_Adaptor_F <400> 191 acactctttc cctacacgac gctcttccga tctagtgttc agtctccgtg aacgttc 57 <210> 192 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> hDNMT1_Adaptor_R <400> 192 gtgactggag ttcagacgtg tgctcttccg atcttcctta gcagcttcct cctcctt 57 <210> 193 <211> 56 <212> DNA <213> Artificial Sequence <220> <223> hCCR5_Adaptor_F <400> 193 acactctttc cctacacgac gctcttccga tctaacagtt tgcattcatg gagggc 56 <210> 194 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> hCCR 5_Adaptor_R <400> 194 gtgactggag ttcagacgtg tgctcttccg atctagttta tcaggatgag gatgacc 57 <210> 195 <211> 59 <212> DNA <213> Artificial Sequence <220> <223> hFANCF_Adaptor_F <400> 195 acactctttc cctacacgac gctctt ccga tctcactacc tacgtcagca cctgggacc 59 <210> 196 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> hFANCF_Adaptor_R <400> 196 gtgactggag ttcagacgtg tgctcttccg atctggaagt tcgctaatcc cggaactgga 60 60 <210> 197 <211> 63 <212> DNA <213 >Artificial Sequence <220> < 223> hGRIN2B_Adaptor_F <400> 197 acactctttc cctacacgac gctcttccga tctctctcat tctgcagagc aaataccaga 60 gat 63 <210> 198 <211> 64 <212> DNA <213> Artificial Sequence <220> <223> hGRIN2B_Adaptor_ R<400> 198 gtgactggag ttcagacgtg tgctcttccg atctcctgca aacacaaaga aagagcatgt 60 taaa 64 <210> 199 <211> 58 <212> DNA <213> Artificial Sequence <220> <223> hEMX1_Adaptor_F <400> 199 acactctttc cctacacgac gctcttccga tctggcctcc tgagtttctc atctgtgc 58 <210> 200 <211> 57 <212> DNA <213> Artificial Sequence <220> <223> hEMX1_Adaptor_R <400> 200 gtgactggag ttcagacgtg tgctctt ccg atctcctgct tcgtggcaat gcgccac 57 <210> 201 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 (wt-crRNA) CRISPR-Cas12a (AsCpf1) Target DNA <400> 201 ttttgtgggc aacatgctgg tcatcctc 28 <210> 202 <211> 43 <212> RNA < 213> Artificial Sequence <220> <223> hCCR5 (wt-crRNA) CRISPR-Cas12a (AsCpf1) Guide RNA <400> 202 aauuucuacu cuuguagaug ugggcaacau gcuggucauc cuc 43 <210> 203 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hCCR5 (chimeric DNA-RNA)CRISPR-Cas12a(AsCpf1)Target DNA <400> 203 ttttgtgggc aacatgctgg tcatcctc 28 <210> 204 <211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hCCR5 (chimeric DNA-RNA)CRISPR-Cas12a(AsCpf1) Guide Chimeric DNA-RNA <220> <223> Chimeric DNA-RNA(TCATCCTC) <400> 204 aauuucuacu cuuguagaug ugggcaacau gcuggtcatc ctc 43 <210> 205 <211 > 32 <212> DNA <213> Artificial Sequence <220> <223> hBRAF (wt-crRNA)CRISPR-Cas12a (AsCpf1) Target DNA <400> 205 ttttttttgg tctagctaca gagaaatctc ga 32 <210> 206 <211> 43 <212 > RNA <213> Artificial Sequence <220> <223> hBRAF (wt-crRNA) CRISPR-Cas12a (AsCpf1) Guid RNA <400> 206 aauuucuacu cuuguagaug gucuagcuac agagaaaucu cga 43 <210> 207 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> hBRAF (chimeric DNA-RNACRISPR-Cas12a (AsCpf1) Target DNA <400> 207 ttttttttgg tctagctaca gagaaatctc ga 32 <210> 208 < 211> 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hBRAF (chimeric DNA-RNACRISPR-Cas12a (AsCpf1) Chimeric DNA-RNA <220> <223> Chimeric DNA-RNA (AATCTCGA) <400> 208 aauuucuacu cuuguagaug gucuagcuac agagaaatct cga 43 <210> 209 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hVEGFA (wt-crRNA)CRISPR-Cas12a(AsCpf1) Target DNA <400> 209 tttctgtcct cagtggtcc c aggctgca 28 <210> 210 <211> 43 <212> RNA <213> Artificial Sequence <220> <223> hVEGFA (wt-crRNA)CRISPR-Cas12a (AsCpf1) Guide RNA <400> 210 aauuucuacu cuuguagauu guccucagug gucccaggcu gca 43 <210 > 211 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> hVEGFA (chimeric DNA-RNA) CRISPR-Cas12a (AsCpf1) Target DNA <400> 211 tttctgtcct cagtggtccc aggctgca 28 <210> 212 <211 > 43 <212> DNA_RNA <213> Artificial Sequence <220> <223> hVEGFA (chimeric DNA-RNA)CRISPR-Cas12a(AsCpf1) Guide Chimeric DNA-RNA <220> <223> Chimeric DNA-RNA(AGGCTGCA)<400 > 212 aauuucuacu cuuguagauu guccucagug gucccaggct gca 43

Claims (14)

Cpf1 protein or DNA encoding it; and
A chimeric DNA-RNA guide containing a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same,
The chimeric (chimeric) DNA-RNA guide is a composition for genome editing, in which 6 to 10 DNAs are located at the 3' end. The method of claim 1, wherein the guide is modified with a phosphate whose 3'-end 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 dielectric correction that is modified by any one selected from the group consisting of hydroxyl and combinations thereof. delete delete The composition for genome editing according to claim 1, further comprising SpCas9 nickase (D10A) or dead SpCas9 nickase (D10A or H840A). A method for producing a transformant comprising the step of introducing the genome editing composition of claim 1 into isolated cells of a non-human organism or into a non-human organism. Inactive Cpf1 (dCpf1) protein or DNA encoding it; and
A chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same,
The chimeric (chimeric) DNA-RNA guide is a composition for inhibiting gene expression, in which 6 to 10 DNAs are located at the 3' end. The composition for inhibiting gene expression according to claim 7, wherein the 3'-end of the guide is modified with phosphorothioate. delete The composition for inhibiting gene expression according to claim 7, wherein the composition further comprises SpCas9 nickase (D10A). Cpf1 protein or DNA encoding it; And a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same,
Wherein the chimeric DNA-RNA guide is located at the 3' end of 6 to 10 DNA, the pharmaceutical composition. Cpf1 protein or DNA encoding it; And a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same,
The chimeric DNA-RNA guide is for preventing or treating a genetic disease, a non-genetic disease, a viral infection, a bacterial infection, cancer, or an autoimmune disease, in which 6 to 10 DNAs are located at the 3' end. pharmaceutical composition. Cpf1 protein or DNA encoding it; and a chimeric DNA-RNA guide comprising a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same;
introducing into an isolated cell of a non-human organism or an in vivo cell of a non-human organism that contains and expresses a DNA molecule having a target sequence and encoding a gene product;
The chimeric DNA-RNA guide is a method of altering the expression of a gene product, in which 6 to 10 DNAs are located at the 3' end. Cpf1 protein or DNA encoding it; and
A chimeric DNA-RNA guide containing a nucleotide sequence hybridizable with a target nucleotide sequence or a DNA encoding the same,
Wherein the chimeric DNA-RNA guide is located at the 3' end of 6 to 10 DNA, gene detection composition.

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