KR101908593B1 - Guide RNA sequences for CFTR gene deletion, T84 cell having mutated CFTR protein and use thereof - Google Patents

Abstract

A polynucleotide complementary to the CFTR gene according to an embodiment, a vector comprising the polynucleotide or a complementary polynucleotide thereof, a composition for mutating a nucleic acid sequence encoding a CFTR polypeptide in a genome of a cell containing the polynucleotide or a complementary polynucleotide thereof, And methods of using the same. According to this, it is possible to mutate the nucleic acid sequence encoding the CFTR polypeptide in the cell or in the individual's genome, to inhibit the expression or activity of the CFTR polypeptide, and to screen the drug for treating CFTR mediated diseases.

Description

CFTR gene deletion induction guide RNA, T84 cell with CFTR mutation and its use {Guide RNA sequences for CFTR gene deletion, T84 cell having mutated CFTR protein and use thereof}

A polynucleotide complementary to a CFTR gene, a vector, a composition for mutating a nucleic acid sequence encoding a CFTR polypeptide in a genome of a cell containing the same, a cell having a CFTR gene mutation using the polynucleotide, and a method using the same.

Genome editing is a technique that can introduce mutations that are targeted to the genomic sequences of animal and plant cells, including human cells, that knock-out or knock-in specific genes, Quot; refers to introducing a mutation into a non-coding DNA sequence that does not generate it. Genetic scissors refers to an enzyme that binds to a gene and cleaves a specific DNA site, or a genome editing technique using the enzyme. Using gene scissors, stem cells or somatic cells can be used in a variety of fields such as mutation correction and cancer cell therapy that cause genetic diseases. As gene scissors, ZFN (zinc finger nuclease) as a first generation gene scissors, TALEN (transcriptional activator-like effector nuclease) as a second generation gene scissors, CRISPR / Cas as a third generation gene scissors, and CRISPR / Cpf1 as a fourth generation gene scissors Is known. Cas9 nuclease and proper guide By transferring RNA into the cell, the cell's genome can be cleaved at the desired location, removing the existing gene and inserting the new gene. When cleaving a specific DNA using gene scissors, the Cas9 nuclease cleaves the DNA target sequence specified by the sequence of the guide RNA. Methods for editing genomes using gene scissors are known in numerous publications including Korean National Publication No. 10-2015-0101478 (2015.09.03).

Cystic fibrosis trans-membrane conductance regulators (cystic fibrosis conductance transmembrane regulator: CFTR ) is a kind of delivery system that controls the transport of anions, HCO3 in the respiratory and gastrointestinal epithelial ^cells play an important role in the secretion and secretion regulation of epithelial secretions And regulates the activity of other ion channels and proteins, as well as an anion flow through the membrane. The disease mediated by CFTR is not only cystic fibrosis but also chronic obstructive pulmonary disease (COPD), dry eye disease or Sjogren's syndrome.

The most common genetic variation among the genetic mutations of the CFTR protein is CFTR, i.e.,? F508-CFTR, in which phenylalanine, the 508th amino acid of CFTR, is deleted (William J. Welch / Seminars in Cell & Developmental Biology 15 ). In order to treat cystic fibrosis, studies on drugs that increase the cell membrane activity or expression of? F508-CFTR protein have been continuously carried out. However, in order to screen drugs having excellent therapeutic effects, The research and development on cell lines is insufficient. Therefore, as a preliminary step for preparing a cell line expressing the? F508-CFTR protein by inhibiting wild-type CFTR protein expression, It is necessary to develop a guide RNA targeting the CFTR gene.

One aspect provides a polynucleotide comprising two or more contiguous nucleotides complementary to a nucleic acid sequence encoding a CFTR polypeptide.

Another aspect provides a vector comprising the polynucleotide or a complementary polynucleotide thereof.

Another aspect provides a composition for altering a nucleic acid sequence encoding a CFTR polypeptide in the genome of a cell.

Another aspect provides a method of mutating a nucleic acid sequence encoding a CFTR polypeptide in the genome of a cell.

Another aspect provides cells in which the nucleic acid sequence encoding the CFTR polypeptide has been mutated.

Another aspect provides a method of screening a test compound that modulates CFTR polypeptide activity.

One aspect provides a polynucleotide comprising two or more contiguous nucleotides complementary to a nucleic acid sequence encoding a CFTR polypeptide.

Cystic fibrosis trans-membrane conductance regulators (cystic fibrosis conductance transmembrane regulator: CFTR ) is a kind of delivery system that controls the transport of anions, HCO3 in the respiratory and gastrointestinal epithelial ^cells play an important role in the secretion and secretion regulation of epithelial secretions And regulates the activity of other ion channels and proteins, as well as an anion flow through the membrane. Mutations in the CFTR gene may result in a lack of sufficient CFTR on the epithelial surface and / or defects in the ion channel activity of the CFTR present on the epithelial surface.

The CFTR polypeptide may be a wild type CFTR polypeptide. The human CFTR polypeptide can be obtained from GenBank Accession No. It may contain an amino acid sequence registered with NP_000483.3. The CFTR polypeptide may comprise the amino acid sequence of SEQ ID NO: 5. The nucleic acid sequence encoding the CFTR polypeptide is described in GenBank Accession No. < RTI ID = 0.0 > No. < / RTI > Or a nucleic acid sequence encoding an amino acid sequence registered with NP_000483.3. The nucleic acid sequence encoding the CFTR polypeptide can be found, for example, in GenBank Accession No. < RTI ID = 0.0 > It may contain a nucleic acid sequence registered with NM_000492.3. The nucleic acid sequence encoding the CFTR polypeptide may comprise the nucleic acid sequence of SEQ ID NO: 1.

The nucleic acid sequence encoding the CFTR polypeptide may, in the case of a human, comprise a nucleic acid sequence of Exon 1 or Exon 2 in the nucleic acid sequence encoding the CFTR polypeptide. The nucleic acid sequence encoding the CFTR polypeptide can be obtained from GenBank Accession No. < RTI ID = 0.0 > It may contain the nucleic acid sequence of exon 1 or exon 2 among the nucleic acid sequences registered with NM_000492.3. The nucleic acid sequence encoding the CFTR polypeptide can be obtained from GenBank Accession No. < RTI ID = 0.0 > (Target 1) 129 to 148th nucleotide, (target 2) 119th to 138th nucleotide, or (target 3) 185 to 204th nucleotide from the 5'end of the nucleic acid sequence registered with NM_000492.3.

The nucleic acid sequence encoding the CFTR polypeptide is selected from the group consisting of the 129th to 148th nucleotides from the 5 'end of SEQ ID NO: 1 (target 1), the 119th to 138th nucleotides from the (target 2) nucleotide sequence of SEQ ID NO: 1, 3) 185 to 204 < th > nucleotides.

The polynucleotide may be a guide RNA. The guide RNA is RNA specific to the target sequence, which can be coupled with a Cas polypeptide to direct Cas polypeptides to the target nucleic acid sequence. The polynucleotide may be a single-chain guide RNA (sgRNA). The guide RNA may be one that contains two RNAs, namely, a CRIS (CRISPR RNA) and a tracrRNA (trans-activating crRNA), or a fusion of a major part of a crRNA (CRISPR RNA) and a tracrRNA have.

The polynucleotide may be selected from the group consisting of the nucleic acid sequences of SEQ ID NOS: 2, 3 and 4. The nucleic acid sequence of SEQ ID NO. It may be complementary to two or more consecutive sequences in exon 1 among the nucleic acid sequences registered with NM_000492.3. The nucleic acid sequence of SEQ ID NO: 2 may be complementary to two or more consecutive nucleotides in the 129th to 148th nucleotides from the 5'-end of SEQ ID NO: 1 in the nucleotide sequence of SEQ ID NO: 1 (target 1). The nucleic acid sequence of SEQ ID NO: 2 may be 5'-GAGGCGACCUCUGCAUGGUC-3 '.

The nucleic acid sequence of SEQ ID NO. It may be complementary to two or more consecutive sequences in exon 1 among the nucleic acid sequences registered with NM_000492.3. The nucleic acid sequence of SEQ ID NO: 3 may be complementary to two or more consecutive nucleotides in the 119th to 138th nucleotides from the 5 'end of SEQ ID NO: 1 in the nucleotide sequence of SEQ ID NO: 1 (target 2). The nucleic acid sequence of SEQ ID NO: 3 may be 5'-CUGCAUGGUCUCUCGGGCGC-3 '.

The nucleic acid sequence of SEQ ID NO. It may be complementary to two or more contiguous sequences in exon 2 among the nucleic acid sequences registered with NM_000492.3. The nucleic acid sequence of SEQ ID NO: 4 may be complementary to two or more consecutive nucleotides within the 185 to 204th nucleotide from the 5 'end of SEQ ID NO: 1 in the polynucleotide consisting of the nucleic acid sequence of SEQ ID NO: The nucleic acid sequence of SEQ ID NO: 4 may be 5'-CAAAAUUGGUCUGGUCCAGC-3 '.

The polynucleotide may further comprise RNA, DNA, PNA, or a combination thereof. For example, it may further comprise DNA encoding the guide RNA. The polynucleotide may be chemically modified.

The nucleic acid sequence may comprise a protospacer adjacent motif (PAM) or a complementary sequence thereof. PAM is a region rich in guanine (G), which serves as a target for target sequences that undergo genetic editing. In other words, the guide RNA finds a cleaved target sequence (proto spacer) adjacent to the PAM. The PAM may comprise a nucleic acid sequence selected from the group consisting of 5'-AGG-3 ', 5'-GGG-3', 5'-CGG-3 'and 5'-TGG-3'.

The polynucleotide may be 10 nucleotides to 40 nucleotides in length. The polynucleotide may be a polynucleotide having a length of, for example, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 nucleotides.

The polynucleotide may be a component of a programmable nuclease. Genetic scissors are all types of nuclease that can recognize and cleave specific positions on the genome. The gene scissors are, for example, zinc finger nuclease (ZFN), transcriptional activator-like effector nuclease (TALEN), CRISPR / Cas, or CRISPR / Cpf1.

The CRISPR / Cas is also referred to as RNA-guide engineered nuclease (RGEN). The CRISPR / Cas consists of a Cas polypeptide that cleaves the target sequence and RNA that binds complementarily to the sequence to be cleaved. The polynucleotide may be an RNA constituting CRISPR / Cas. The polynucleotide may be a guide RNA constituting CRISPR / Cas.

The programmed Cas polypeptide induces a double strand break (DSB) in the target sequence near the PAM. The double strand breaks are restored by non-homologous end-joining (NHEJ), which is incomplete to form an insert / indel region or to form an insert / It can cause frame-shift mutation.

The polynucleotide induces double strand breaks in the target sequence of the CFTR gene near PAM. This double-strand break is restored by non-homologous end joining, which can result in insertion / deletion sites in the nucleic acid sequence encoding the CFTR polypeptide, resulting in lattice mobility shifts. The mutated CFTR gene can be increased or suppressed in its expression.

Another aspect provides a vector comprising a polynucleotide or a complementary polynucleotide thereof according to one aspect.

The vector may be introduced into a cell or an individual to produce a CFTR guide RNA. The vector may be introduced into a cell or an individual to express a CFTR guide RNA and / or a Cas polypeptide and induce lattice mobility by forming an insertion / deletion site in the nucleic acid sequence encoding the CFTR polypeptide in the cell or the individual.

Such vectors can be constructed through a variety of methods known in the art (Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001 and Cong et al Science.

The vector may be a vector for cloning or a vector for expression. The vector may be a viral vector, a plasmid vector, or an agrobacterium vector.

The vector may be constructed with prokaryotic or eukaryotic cells as hosts. When the vector is an expression vector and the prokaryotic cell is used as a host, a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pL? Promoter, pR? Promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, and T7 promoter), a ribosome binding site for initiation of detoxification, and a transcription / translation termination sequence. When the vector is a vector for expression and eukaryotic cells are used as a host, a promoter derived from the genome of a mammalian cell (for example, a metallothionine promoter) or a mammalian virus (for example, , The late adenovirus promoter, the vaccinia virus 7.5K promoter, the SV40 promoter, the cytomegalovirus promoter, and the tk promoter of HSV), and may have a polyadenylation sequence as a transcription termination sequence. On the other hand, the vector may be a plasmid (for example, pSC101, ColE1, pBR322, pUC, pUC8 / 9, pHC79, pUC19 and pET etc.), a phage (for example, λgt4 · λB, λ- And M13) or a virus (e.g., SV40 or the like). The vector may comprise a CBh promoter, a U6 promoter, or a combination thereof.

 The vector may comprise an antibiotic resistance gene. The antibiotic resistance gene may be an ampicillin resistance gene, a puromycin resistance gene, chloramphenicol, a blasticidin resistance gene, or a combination thereof.

Another aspect provides a composition for altering a nucleic acid sequence encoding a CFTR polypeptide in a cell's genome, comprising a polynucleotide according to one aspect, a vector according to one aspect, or a combination thereof.

The nucleic acid sequences encoding the polynucleotides, vectors, CFTR polypeptides, and CFTR polypeptides are as described above.

The cell may be derived from an organ that performs a secretory function. The cells may be selected from the group consisting of lung, bronchial, liver, pancreas, bile duct, bowel, intestine, kidney, ureter, bladder, urethra, testes, urethra, prostate, bladder, penis, anus, ureter, ovary, uterus, vagina, It may be derived. The cell may be an epithelial cell, a tumor cell, a stem cell, a vascular endothelial cell, a white blood cell, an immune cell, a germ cell, a fibroblast, a muscle cell, a bone marrow cell, a epidermal cell, an osteoblast, a nerve cell and a combination thereof. The cell may be, for example, an epithelial cell of an organ that performs a secretory function.

The mutation may be deletion, substitution, insertion, insertion / deletion of one or more nucleotides or lattice mobility mutation thereof. The "mutation" can be used interchangeably with "mutation" or " mutation ". The mutation means any modification in which the expression, activity or function of the CFTR polypeptide is increased, lost or attenuated by altering the nucleic acid sequence encoding the CFTR polypeptide in the genome of the cell.

Said mutation comprising a polynucleotide comprising a nucleic acid sequence encoding said CFTR polypeptide, a polynucleotide comprising a nucleic acid sequence encoding exon 1 or exon 2 in a nucleic acid sequence encoding a CFTR polypeptide, A polynucleotide comprising a nucleic acid sequence registered with NM_000492.3; A polynucleotide comprising a nucleic acid sequence of exon 1 or exon 2, a polynucleotide consisting of a nucleic acid sequence encoding a polypeptide consisting of the amino acid sequence of SEQ ID NO: 5, or a polynucleotide comprising a nucleic acid sequence of SEQ ID NO: Deletion, substitution, insertion, insertion / deletion, or lattice mobility mutation of one or more nucleotides in the vicinity of the prospective spacer adjacent motif (PAM) or in the upstream region thereof in the polynucleotide made.

The mutation may be one in which all or some of the mutations are present in a pair of homologous chromosomes (alleles). The mutation may be, for example, a nucleotide 129 to 148, a nucleotide 119 to 138 (target 2), or a nucleotide 119 to 138 (target 3) from the 5 'end of SEQ ID NO: 1 in a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: ) Deletion, substitution, insertion, insertion / deletion, or lattice mobility mutation of one or more nucleotides at 185 to 204 nucleotides. For example, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, nucleotides 128 to 144 are deleted from the 5 'end of SEQ ID NO: 1, guanine (G) is inserted at the 128 th position, , And cytosine (C), guanine (G) and adenine (A) at the 144th position, respectively. For example, in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1, the nucleotides 124 to 138 from the 5 'end of SEQ ID NO: 1 are deleted and the nucleotides at positions 124, 125, 126, 127, (A), cytosine (C), and cytosine (C) at the 134th, 135th, 136th, 137th, and 138th positions, respectively, , Adenine (A), and thymine (T).

The composition may be for in vitro or in vivo administration.

The composition may comprise a composition capable of facilitating introduction of a polynucleotide according to one aspect, a vector according to an aspect, or a combination thereof into a cell, and instructions for preparation or introduction into a cell .

The composition may be a pharmaceutical composition. The pharmaceutical composition may be one for the prevention or treatment of CFTR mediated diseases. The pharmaceutical composition may comprise a pharmaceutically acceptable carrier. The carrier is used to mean an excipient, diluent or adjuvant. The carrier may be, for example, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Buffers such as saline, water, physiological saline, PBS, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. The composition may be a filler, an anticoagulant, a lubricant, a wetting agent, a flavoring agent, an emulsifying agent, a preservative, or a combination thereof.

The pharmaceutical composition may be prepared in any formulation according to a conventional method. The composition may be formulated, for example, in an oral dosage form (e.g., powder, tablet, capsule, syrup, pellet, or granule) or a parenteral formulation (e.g., an injection). In addition, the composition may be formulated into a whole-body or local formulation. The pharmaceutical composition may be administered orally, intravenously, intramuscularly, orally, transdermally, mucosally, intranasally, intratracheally, subcutaneously, or a combination thereof.

The pharmaceutical composition may comprise an effective amount of a polynucleotide according to one aspect, a vector according to an aspect, or a combination thereof. The term "effective amount " refers to an amount sufficient to exhibit the effect of prevention or treatment when administered to a subject in need of such prevention or treatment. The effective amount may be appropriately selected depending on the cell or individual selected by a person skilled in the art. The severity of the disease, the age, weight, health, sex, sensitivity of the patient to the drug, time of administration, route of administration and rate of excretion, duration of treatment, elements including drugs used in combination with or co- May be determined according to well known factors.

The composition may further comprise a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide.

The CRISPR / Cas consists of a Cas polypeptide that cleaves the target sequence and RNA that binds complementarily to the sequence to be cleaved. The Cas polypeptide may be one that forms an endonuclease or nick as a component of the CRISPR / Cas.

The Cas polypeptide may be a Cas polypeptide derived from a microorganism. Such Cas polypeptides include, for example, Streptococcus spp. (E.g. , Streptococcus pyogens ), Nepheline spp . (Such as Neisseria sp. meningitidis , Pasteurella sp. (e.g., Pasteurella multocida ), francicella genus (e.g., Francisella novicida ), or campylobacter genus (e. g., Campylobacter jejuni ). < / RTI > The Cas polypeptide may be obtained from GenBank Accession No. < RTI ID = 0.0 >Lt; RTI ID = 0.0 > Q99ZW2. ≪ / RTI > The Cas polypeptide may be obtained from GenBank Accession No. < RTI ID = 0.0 > Or an amino acid sequence encoded by the nucleic acid sequence of KT031982.1. The Cas polypeptide derived from the microorganism may be codon-optimized and humanized.

The Cas polypeptide may be a recombinant protein.

The Cas polypeptide may be a wild-type Cas polypeptide, or a mutant Cas polypeptide. The mutant Cas polypeptide may be, for example, a Cas polypeptide variant in which the catalytic aspartate residue is changed to another amino acid (for example, alanine). The Cas polypeptide may be an activated Cas polypeptide, an inactivated Cas (dCas) polypeptide, or a Casase nickase. The Cas polypeptide may be a Cas5 polypeptide, a Cas7 polypeptide, a Cas8 polypeptide, a Cas9 polypeptide, or a Cpf1 polypeptide. The Cas9 polypeptide may be, for example, a wild type Cas9, an inactivated Cas9 (dCas9) polypeptide, or a Cas9 nicase. The inactivated Cas9 polypeptide may be an RFN (RNA-guided FokI Nuclease) or a dCas9 polypeptide in which a FokI nuclease domain is linked to a dCas9 polypeptide, or a transcription activator or a repressor domain is connected to the dCas9 polypeptide And the Cas9 niche may be D10A Cas9 or H840A Cas9, but is not limited thereto.

The composition may be to increase or inhibit the expression, activity or function of the CFTR polypeptide. The expression, activity, or function is inhibited when the polypeptide activity in a cell having a given genetic modification (e. G., A cell in which the nucleic acid sequence encoding the CFTR polypeptide is mutated) does not have the same type of genetic modification At least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60% of the activity of the polypeptide in the cell (negative control, e.g., , About 70% or more, or about 100% or more. The increased expression, activity or function indicates that the polypeptide activity in a cell with a given genetic modification (e. G., A cell in which the nucleic acid sequence encoding the CFTR polypeptide has been mutated) does not have the same type of genetic modification At least about 10%, at least about 15%, at least about 20%, at least about 30%, at least about 50%, at least about 60% of the activity of the polypeptide in the cell (negative control, e.g., , About 70% or more, or about 100% or more. The CFTR polypeptide activity or function means activity or function as a channel for an anion, for example, Cl < ^- > in a cell membrane, for example.

Another aspect provides a method of mutating a nucleic acid sequence encoding a CFTR polypeptide in a cell's genome, comprising incubating the cell and a polynucleotide according to an embodiment, a vector according to an aspect, or a combination thereof.

The polynucleotides, vectors, CFTR polypeptides, nucleic acid sequences encoding mutations and CFTR polypeptides, and the increase or inhibition of the expression, activity or function of CFTR polypeptides are as described above.

The incubation may be by culturing the cells with a polynucleotide according to one aspect, a vector according to an aspect, or a combination thereof. The incubation may be performed under conditions in which the cell is viable. Said viable condition may be a condition allowing it to be in a proliferative condition or a condition to be in a resting state. The viable condition may suitably control CO ₂ , temperature, humidity, and incubation time.

The incubation may be by introducing a polynucleotide according to an aspect, a vector according to an aspect, or a combination thereof into cells from outside. The introduction into the cell may be carried out using a method known in the art, and may be a step of infecting the cell by transfection or transduction. Transfection may be by calcium phosphate-DNA coprecipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, liposomal fusion, lipofectamine and protoplast fusion .

The incubation can be performed in vitro or in vivo.

The method may further comprise incubating the cell with a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide. This step may be performed concurrently with, prior to, or after incubating the polynucleotide according to one aspect, the vector according to an aspect, or a combination thereof.

The method may be to increase or inhibit CFTR polypeptide expression relative to negative control by mutating the nucleic acid sequence. According to the method, the expression, activity or function of the CFTR polypeptide can be increased or inhibited relative to the negative control.

Another aspect provides cells in which the nucleic acid sequence encoding the CFTR polypeptide has been mutated.

The cell may be derived from a human. The cell may be derived from an organ that performs a secretory function. The cells may be selected from the group consisting of lung, bronchial, liver, pancreas, bile duct, bowel, intestine, kidney, ureter, bladder, urethra, testes, urethra, prostate, bladder, penis, anus, ureter, ovary, uterus, vagina, It may be derived. The cell may be immortalized. The cell may be a cell line. The cell may be a tumor cell. The cells may be cells derived from human colon tumors (e.g. T84), cells derived from the colon epithelium (e.g., CACO-2, HT29), cells derived from the bronchial epithelium (e.g. Calu-3) (E.g., MDCK), nasal epithelial cells, pancreatic ductal epithelial cells (e.g., CAPAN, PANC).

The mutation of the nucleic acid sequence encoding the CFTR polypeptide may be accomplished by a polynucleotide according to one aspect, a vector according to an aspect, a composition for altering the nucleic acid sequence encoding the CFTR polypeptide in the genome of the cell according to one aspect, By mutating the nucleic acid sequence encoding the CFTR polypeptide from the genome of the cell according to the method of the present invention, or a combination thereof.

The cells were obtained from GenBank Accession No. Or a mutation in the nucleic acid sequence of exon 1 or exon 2 among the nucleic acid sequences registered with NM_000492.3. The cell may be, for example, a nucleotide 129 to 148, a nucleotide 119 to 138 (target 3), or a nucleotide sequence of 185 to 204 (target 3) from the 5 'terminus in the polynucleotide consisting of the nucleotide sequence of SEQ ID NO: Lt; RTI ID = 0.0 > nucleotides. ≪ / RTI >

The cell may be one having deletion, substitution, insertion, insertion / deletion, or lattice mobility mutation of one or more nucleotides in the vicinity of the prospective spacer adjacent motif (PAM) or upstream thereof.

The cell may be one whose expression, activity, or function of the wild-type CFTR polypeptide is increased or suppressed.

The polynucleotides, vectors, CFTR polypeptides, nucleic acid sequences encoding mutations and CFTR polypeptides, and the increase or inhibition of the expression, activity or function of CFTR polypeptides are as described above.

The cells may be those with increased expression of? F508-CFTR polypeptide. The cell may have an increased expression of CFTR (ΔF508-CFTR) polypeptide in which the phenylalanine, which is the 508th amino acid from the N terminus of SEQ ID NO: 5, is deleted in the polypeptide consisting of the amino acid sequence of SEQ ID NO: 5 compared to the negative control. The? F508-CFTR polypeptide may comprise the amino acid sequence of SEQ ID NO: 17.

The ΔF508-CFTR polypeptide is the most common genetic variation among genetic mutations of the CFTR protein, and refers to a CFTR protein in which phenylalanine, the 508th amino acid in the amino acid sequence of CFTR, is deleted. Isoleucine, which is the 507th amino acid in the amino acid sequence of CFTR, is encoded by TAG and phenylalanine, which is the 508th amino acid in the amino acid sequence of CFTR, is encoded by AAA. The ΔF508-CFTR protein has TAA as a codon that encodes the 507th and 508th amino acids as a result of deletion of the first AA among G and AAA in the TAG. The TAA encodes isoleucine, but phenylalanine, the 508th amino acid in the amino acid sequence of the wild-type CFTR, is deleted.

The [Delta] F508-CFTR protein is structurally misfolded, i.e., folding defective protein. The passage does not function ^- such △ F508-CFTR protein because protein folding phenomenon due to the loss of a phenylalanine abnormally up the cell does not pass through the normal protein function test Cl due to the difficulty to reach the cell membrane. However, once the △ F508-CFTR protein reaches the cell membrane, it can function as a Cl ^- channel.

The increased expression of the ΔF508-CFTR polypeptide may be due to the inclusion of at least one exogenous gene having a nucleic acid sequence encoding the ΔF508-CFTR polypeptide. The exogenous gene refers to a gene that is introduced into the cell from the outside, and the introduced gene may be homologous or heterologous to the host cell to be introduced. The exogenous gene may be integrated into the genome of the cell, or may be present independently of it. The introduction into the cell can be carried out by methods known in the art, and it goes as described above.

The increased expression of the ΔF508-CFTR polypeptide can be obtained by introducing a gene having a nucleic acid sequence encoding ΔF508-CFTR polypeptide into a cell using a viral vector system. Such viral vectors include retroviruses, adenoviruses, adeno-associated viruses (AAV), and other viruses that include herpes simplex virus (HSV) and poxviruses .

The increased expression of the? F508-CFTR polypeptide may be by introducing into the cell a gene having a nucleic acid sequence encoding the? F508-CFTR polypeptide by a retroviral vector.

Since target cells are not infected with retrovirus when they are in the G0 phase, cells can be prestimulated and induced into the cell cycle.

The retroviral vector may be a recombinant retroviral vector. The retroviral vector may be a replication-defective recombinant retroviral vector. These vectors are not self-replicating and non-pathogenic in infected cells because of their poor replication potential. Retroviruses in which vectors are packaged can infiltrate host cells such as vertebrate cells and mammalian cells and introduce exogenous genes inserted into vectors into chromosomal DNA stably.

The retroviral vector may be a lentiviral vector. Lentiviruses enable active introduction of lentiviral capsids into nucleopores or nuclear membranes because they enter the nucleus of the cells through nuclear pores and can infect not only mitotic cells but also mitotic cells.

The lentiviral vector may further comprise a packaging vector, and / or an envelope vector. The packaging vector may be one that expresses a gag-pol protein. The envelope vector may be one that expresses an env protein capable of binding a specific cell surface receptor protein.

The cell may be one in which the expression, activity, or function of the wild-type CFTR polypeptide is suppressed and at the same time the ΔF508-CFTR polypeptide is expressed.

The cell may be that of a cell line having the accession number KCLRF-BP-00388.

In another aspect, there is provided a method comprising: incubating a cell and a test compound according to an aspect; Measuring CFTR polypeptide activity in the incubated cells; And screening a test compound that has increased or decreased the measured CFTR polypeptide activity relative to a negative control, comprising screening a test compound that modulates CFTR polypeptide activity.

The method may comprise incubating the cells with the test compound according to an aspect. Cells and incubation are as described above.

The test compound means a candidate substance expected to increase CFTR polypeptide activity, expression or function. The CFTR polypeptide activity or function means activity or function as a channel for an anion, for example, Cl < ^- > in a cell membrane, for example.

The CFTR may be that the polypeptide is a wild type CFTR polypeptide or a mutant CFTR polypeptide. The mutant CFTR polypeptide may be a? F508-CFTR polypeptide.

The method may comprise measuring the CFTR polypeptide activity. Methods for measuring the CFTR polypeptide activity include, but are not limited to, halide ion quenching, Cl ^- / HCO ₃ ^- exchange by intracellular alkalinization, pH change, Cl ^- current measurement of CFTR using patch ^- , Confirming the post-Golgi fully-glycosylated form of the CFTR polypeptide (band C) through the ER core glycosylated form of CFTR polypeptide (band B) or Golgi in the endoplasmic reticulum Western blot, immunofluorescence staining and PCR, and the like. The method of measuring the CFTR polypeptide activity may be a method of measuring polypeptide activity known in the art.

The method may comprise selecting a test compound that has increased or decreased the measured CFTR polypeptide activity relative to the negative control. The method may include measuring CFTR polypeptide activity in cells that are not incubated with the test compound and comparing the measured CFTR polypeptide activity to CFTR polypeptide activity in the incubated cells with the test compound.

When a test compound that is expected to increase CFTR polypeptide activity with a cell according to one aspect is cultured and the level of CFTR polypeptide activity or expression is increased in cells treated with the test compound as compared to cells not treated with the test compound, It can be judged that the test compound increases CFTR polypeptide activity.

The method may be for screening a pharmaceutical composition for the prevention or treatment of CFTR gene or CFTR polypeptide mediated diseases. The screened test compounds may be used for the treatment of cystic fibrosis, chronic obstructive pulmonary disease (COPD), dry eye disease, bronchitis, emphysema, bronchiectasis, asthma, sinusitis, Sjogren's syndrome, A candidate compound for the prophylaxis or treatment of a disease selected from the group consisting of Alzheimer's disease, Type II diabetes, Parkinson's disease, and Creutzfeldt-Jakob disease.

The term "prevention" refers to any action that inhibits the development of the disease or delays its onset by administration of the test compound. The term " treatment "refers to any action that improves or alters the symptom of the disease by the administration of the test compound.

When a test compound that is expected to increase CFTR polypeptide activity with a cell according to one aspect is cultured and the level of CFTR polypeptide activity or expression is increased in cells treated with the test compound as compared to cells not treated with the test compound, It can be judged that the test compound has an effect of preventing or treating the above-mentioned diseases.

According to one aspect of the present invention, there is provided a polynucleotide complementary to a CFTR gene, a vector, a composition for mutating a nucleic acid sequence encoding a CFTR polypeptide in a genome of a cell containing the same, a cell having a CFTR gene mutation using the same, Or can mutate the nucleic acid sequence encoding the CFTR polypeptide in the genome of the subject, inhibit the expression or activity of the CFTR polypeptide, and can be used to screen for drugs for treating CFTR mediated diseases.

Figure 1A shows the positions of target candidate sequences 1, 2 and 3 on the CFTR gene. Figure IB shows the process of cleaving the target sequence 2 to the guide RNA complementary to the target sequence 2 and Cas9 nuclease. Fig. 1C shows the position at which the mutation occurred and the size of the DNA fragment in targets 1, 2 and 3 on the CFTR gene. Figure 1D shows that Cas9 induces mutation only in the presence of the guide RNA in HEK293T cells, resulting in a 447 bp DNA fragment for target 1, a 457 bp DNA fragment for target 2, and a 394 bp DNA fragment for target 3 . FIG. 1E shows that the guide RNA and Cas9 induce mutations in T84 cells, and DNA fragments are generated.
Figure 2A shows the structure of a surrogate reporter plasmid in which the plasmid contains a constitutively expressed monomeric Red Fluorescent protein (mRFP), a target sequence, and two outgrowth frames of enhanced green fluorescence Protein (enhanced green fluorescent protein: eGFP). Fig. 2B shows, after 3 days of transfection, T84 cells transfected with a plasmid encoding a surrogate reporter plasmid and a CFTR guide RNA, plasmids encoding Cas9 and plasmid encoding Cas9, and T84 transfected with a plasmid encoding CFTR guide RNA In each cell, the degree of expression of RFP and GFP was observed by fluorescence microscope. FIG. 2C shows, after 3 days of transfection, T84 cells transfected with a plasmid encoding a surrogate reporter plasmid and a CFTR guide RNA, and a plasmid encoding Cas9 and plasmid encoding Cas9, and T84 transfected with a plasmid encoding CFTR guide RNA In each cell, the degree of expression of RFP and GFP is represented by FACS. FIG. 2D shows the frequency of mutation in the target 2 region in exon 1 in a group of cells showing RFP-positive and GFP-positive by T7E1 analysis.
Figure 3A shows the DNA sequence of seven clones and the wild type T84 DNA sequence as a control group. FIG. 3B is a Western blot graph showing that the expression of CFTR was completely suppressed at the # 34 clone in which insertion / deletion occurred, and the degree of CFTR expression at the # 22 clone in which genetic editing was not performed. FIG. 3C shows the expression of ΔF508-CFTR in the prepared cell line by Western blotting.
Fig. 4A is a fluorescence image showing the expression of halide-sensitive YFP in T84-ΔF508 cells. 4B shows the degree of YFP expression when T84-ΔF508 cells were treated by treatment with CFTR _inh -172 at 37 ° C, 27 ° C, and 27 ° C, respectively. FIG. 4C shows the degree of YFP expression when T84-ΔF508 cells were treated with VX-809, VX-809, VX-770, and VX-809 and CFTR _inh -172, respectively. Figure 4D shows the activity of the CFTR channel when treated with T84-ΔF508 cells treated at 37 ° C and 27 ° C respectively with VX-809, VX-809 and VX-770, and VX-809 with CFTR _inh -172 As shown in the graph. 4E shows Western blots as to whether band C was formed when T84-ΔF508 cells were reacted at 37 ° C., 27 ° C., and VX-809 treatment, respectively.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these embodiments are intended to illustrate one or more embodiments, and the scope of the present invention is not limited to these embodiments.

Example  One. CFTR  Screening of guide RNA, CFTR  Confirming the reduction of CFTR protein expression in cells using guide RNA, and CFTR  Production of stable cell line with suppressed expression

One. CFTR  Selection of target sites in genes CFTR  Guide RNA Fla Smith production

To remove the CFTR gene, a plasmid encoding the CFTR guide RNA (single-chain guid RNA: sgRNA) was prepared.

PX330-U6 containing the cloning site of BbsI (New England Biolabs, Beverly, MA, USA), CRISPR RNA (crRNA) / trans-activated crRNA chimera, and humanized S. pyogenes Cas9 as a plasmid expressing the guide RNA -Chimeric_BB-CBh-hSpCas9 (Addgene plasmid # 42230) vector was used.

Two exons in the human CFTR gene were selected as target candidates for the guide RNA. The exon encodes an amino acid sequence located at the N-terminus of the CFTR protein.

Using an online tool, a site with a 20 bp target-3 bp protospacer adjacent motif (PAM) sequence in the human CFTR exome was identified. The positions of the three candidate target sequences are shown in Fig. 1A.

The guide RNA for the three target candidate sequences was designed as follows. The 3 ' sequence of the strand complementary to the target sequence has the sequence of the trinucleotide PAM, i.e., 5'-NGG-3 '. Also, for example, the guide RNA was designed such that the 5 'end of the guide RNA contained the AUG (ATG) start codon and paired with two DNA target sites. Referring to FIG. 1B, the 3 'sequence of the strand complementary to the target sequence has the sequence of the trinucleotide PAM, 5'-AGG-3', and the guide RNA contains the AUG start codon Respectively.

5'-gaccatgcagaggtcgcctc-3 ', 5'-gcgcccgagagaccatgcag-3' existing in exon 1 and 5'-gctggaccagaccaattttg-3 'existing in exon 2 were selected as target candidates. (Oligonucleotide sequence complementary to target 1 in exon 1: 5'-GACCATGCAGAGGTCGCCTC-3 '(SEQ ID NO: 6) and 5'-GAGGCGACCTCTGCATGGTC-3' (SEQ ID NO: 7); exon 1 3 '(SEQ ID NO: 8) and 5'-CTGCATGGTCTCTCGGGCGC-3' (SEQ ID NO: 9) and an oligonucleotide sequence complementary to target 3 in exon 2 SEQ ID NO: '-GCTGGACCAGACCAATTTG-3' (SEQ ID NO: 10) and 5'-CAAAATTGGTCTGGTCCAGC-3 '(SEQ ID NO: 11). An oligonucleotide sequence complementary to target 1 in exon 1, an oligonucleotide sequence complementary to target 2, and a complementary oligonucleotide sequence to target 3 in exon 2 were synthesized (Cosmo Genetech, Seoul, South Korea), and T4 polynucleotides And annealed using kinase (New England Biolabs).

In order to phosphorylate and anneal each pair of oligonucleotides, PCR was performed using 20 μl of the PCR reaction mixture. The reaction mixture contained 2 μl of 100 mM forward oligonucleotide, 2 μl of 100 mM reverse oligonucleotide, 2 μl of 10 × T4 ligation buffer, 11 μl of distilled water and 1 μl of T4 PNK. Annealing conditions were set at 37 캜 for 45 min, followed by 95 캜 for 5 min and then ramping to 25 캜 (5 캜). The annealed oligonucleotides were ligated to pX330-U6-Chimeric_BB-CBh-hSpCas9 vector digested with BbsI restriction enzyme using T4 DNA ligase. The vector was designated as pX330-CFTR guide RNA plasmid as the plasmid encoding the CFTR guide RNA. The pX330-CFTR guide RNA plasmid contains pX330-CFTR guide RNA-1 for target 1, X330-CFTR guide RNA-1 for target 2, and pX330-CFTR guide RNA-3 for target 3, respectively.

2. Cell culture, intracellular CFTR  Guide RNA introduction and Immunoblot  Analysis method

HEK293T cells, a human embryonic kidney cell line, and T84, a human colon cancer cell line, were transfected with a plasmid encoding the CFTR guide RNA and the degree of expression was confirmed by GFP and immunoblot.

Cell culture and transfection were carried out in the following manner. HEK293T cells were cultured in DMEM (Dulbecco's modified Eagle's medium) -HG containing 100 units / ml of penicillin, 100 占 퐂 / ml of streptomycin and 10% of fetal bovine serum (FBS) ₂ incubator. T84 cells were cultured in DMEM / F-12 containing 100 units / ml of penicillin, 100 占 퐂 / ml of streptomycin and 5% FBS at 37 占 폚 in a 5% CO ₂ incubator. Cells were dosed one day prior to transfection so that the confluency of the cells in the culture plate was about 70-80%.

HEK293T cells were transfected with plasmids encoding the CFTR guide RNA using lipofectamine. To a 6 well plate in which HEK293T cells were dispensed, 2 쨉 g of the plasmid and 6 쨉 l of lipofectamine were added together with opti-MEM (Invitrogen). T84 cells were transfected with a plasmid encoding the CFTR guide RNA using Lipofectamine 2000. To 6 well plate in which T84 cells were dispensed, 2 占 퐂 of plasmid and 6 占 퐇 of lipofectamine 2000 were added together with opti-MEM (Invitrogen). After 6 hours of transfection, the medium was replaced with medium containing serum.

To determine the efficiency of transfection, the degree of GFP expression was determined after 36 hours of transfection. Fluorescence microscopy images showed that about 80% of HEK293T cells were transfected and about 10% of T84 cells were transfected.

Immunoblot (Western blot) analysis was performed in the following manner. After 48 hours of transfection, cells were harvested and lysed with lysis buffer (1% NP-40, 150 mM NaCl, 1 mM EDTA, 50 mM Tris / HCl, pH 7.4) containing protease inhibitor (Roche Applied Science, Mannheim, Germany) Respectively. The lysed cells were centrifuged at 10,000 g for 10 minutes to remove cell debris and top lysates were obtained. The resulting lysate was mixed with 2x SDS sample buffer and separated on SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 4-12% (w / v). The separated proteins were transferred to a nitrocellulose (NC) membrane and the membrane was reacted with TBST (Tris-buffered saline containing 0.1% Tween 20) containing 5% (w / v) Lt; / RTI > Subsequently, the membrane was reacted with a primary antibody, anti-CFTR M3A7 (Millipore Billerica, MA, USA) and anti-aldolase (Santa Cruz Biotechnology, Santa Cruz, , And enhanced chemiluminescence solution (ECS) (Amersham Biosciences, Piscataway, NJ, USA)).

3. T7E1  analysis

To determine whether mutations occurred in the CFTR gene when using the above-mentioned guide RNA sequence, T7E1 analysis was performed and the mutation frequency was measured. The T7E1 assay method was performed according to methods known in the art (Kim et al. 2009; Ramakrishna et al. 2014).

HEK293T cells and T84 cells were each transfected with a plasmid encoding the CFTR guide RNA. HEK293T cells were transfected with a plasmid encoding the CFTR guide RNA, pX330-CFTR guide RNA-1, X330-CFTR guide RNA-2, and pX330-CFTR guide RNA-3, respectively, using lipofectamine. Three days after transfection, cells were harvested and genomic DNA was isolated using a GeneJET genomic DNA purification kit (Thermo Scientific, Waltham, MA, USA). Exon 1 and exon 2 of the CFTR gene were amplified with genomic DNA specific primers. The primer specific for exon 1 is the forward primer 5'-AATTGGAAGCAAATGACATC-3 '(SEQ ID NO: 12) and the reverse primer 5'-TGAAGAATCATTTACCTGTG-3' (SEQ ID NO: 13). The primers specific for exon 2 are the forward primer 5'-GAGGCTGGACTTCAGTAATTTG-3 '(SEQ ID NO: 14) and the reverse primer 5'-CAAGTAACCCTCCTGCTTCAG-3' (SEQ ID NO: 15).

10 [mu] l of homoduplex PCR amplicon, 2 [mu] l of 10x reaction buffer and distilled water were mixed to make 20 [mu] l of mixture, and the mixture was subjected to step-down annealing conditions Ramping (-2 DEG C / s) from 95 DEG C to 85 DEG C; ramping (-0.25 DEG C / s) from 85 to 25 DEG C; and 1 minute holding at 25 DEG C) Rehybridization to generate homologous double stranded and heterologous double stranded DNA. The double-stranded mixture was digested with T7 endonuclease 1 (New England Biolabs) at 37 ° C for 20 minutes. Negative control groups were used which did not contain the guide RNA. The cleaved product was transferred to 2% agarose gel and subjected to electrophoresis, and the size of the DNA fragment was confirmed by band. The intensity of the bands was measured using MultiGauge v3.1 software (Fuji Photo Film Co. Ltd., Tokyo, Japan) and the frequency of indent variations was calculated using the following equation [variation frequency %) = 100 x (1 - (1-fraction cleaved) ^1/2 )] (Guschin et al. 2010).

Fig. 1C shows the position at which the mutation occurred and the size of the DNA fragment in targets 1, 2 and 3 on the CFTR gene. As a result of the T7E1 analysis, it is predicted that the mismatched regions in the targets 1, 2 and 3 are cleaved, resulting in a DNA fragment of 447 bp or 457 bp in exon 1 and a DNA fragment of 394 bp in exon 2.

Figure 1D shows that Cas9 induces mutation only in the presence of the guide RNA in HEK293T cells, resulting in a 447 bp DNA fragment for target 1, a 457 bp DNA fragment for target 2, and a 394 bp DNA fragment for target 3 . The red arrow indicates the DNA fragment generated by cleavage of the mismatched portion by T7E1. As shown in Fig. 1D, Cas9 modified the target DNA sequence only in the expected position when there was a guide RNA. Below the lane, the frequency of insertion / deletion variation for each target is expressed as a percentage. Insertion / deletion mutations occurred in the target candidates of targets 1, 2 and 3, with target 2 showing the highest frequency variation. The CFTR guide RNA sequence for the target 2 of exon 1 was found to produce the most efficient insert / indel mutation in the endogenous CFTR gene.

T84 cells were transfected with a plasmid encoding CFTR guide RNA using lipofectamine 2000 and the same experiment as described above was performed. Fig. 1E shows the results of production of DNA fragment for Tables 1, 2 and 3 by inducing Cas9 mutation in T84 cells. However, as shown in Fig. 1E, in the case of T84 cells, almost no cells with mutations were observed on the agarose gel as compared with the case of HEK293T cells. From this it can be seen that cells with low transfection efficiency, such as T84 cells, require a different strategy to increase the mutation frequency in the Cas9 system or to obtain only the edited cells.

4. Using a surrogate reporter plasmid, CFTR  Cell selection with gene mutation

Among transfected cells, genome editing induced by nuclease can only occur in some cells. Thus, cells transfected with a surrogate reporter plasmid and having a Cas9-induced mutation were selected using fluorescence-activated cell sorting (FACS).

Substitute reporter plasmids were constructed according to the following principle (Ramakrishna et al. 2014). Oligonucleotides containing the target sequence are synthesized (Cosmo Genetech) and annealed in vitro using a thermocycler (5 min at 95 [deg.] C, ramping down to 25 [deg.] C )). The annealed oligonucleotides are ligated into pRG25, a reporter vector cleaved with restriction enzymes such as EcoRl and BamHl. The vector can modulate the generation of a termination codon in a frame at the target site by altering the frame or changing the direction of the target site.

Figure 2A shows the structure of a surrogate reporter plasmid in which the plasmid contains a constitutively expressed monomeric Red Fluorescent protein (mRFP), a target sequence, and two outgrowth frames of enhanced green fluorescence Protein (enhanced green fluorescent protein: eGFP). RFP is constitutively expressed by the CMV promoter (pCMV), whereas GFP is not expressed out of frame when the sequence of GFP is outside the Cas 9 nuclease activity. When a double strand break (DSB) is introduced into the target sequence by a programmed Cas9 nuclease, the double strand break is restored by NHEJ, which forms an insert / Resulting in a frame-shift mutation. The induced lattice mobility shifts GFP expression. Thus, the emission of GFP means that a mutation has occurred in the target site of the cell.

T84 cells were co-transfected with Lipofectamine 2000 using a surrogate reporter plasmid, a plasmid encoding Cas9, and a plasmid encoding the CFTR guide RNA. 24 hours after transfection, a significant number of cells were observed to express RFP, but few cells expressing GFP. Fig. 2B shows, after 3 days of transfection, T84 cells transfected with a plasmid encoding a surrogate reporter plasmid and a CFTR guide RNA, plasmids encoding Cas9 and plasmid encoding Cas9, and T84 transfected with a plasmid encoding CFTR guide RNA In each cell, the degree of expression of RFP and GFP was observed by fluorescence microscope. As shown in FIG. 2B, over the three days after transfection, the number of cells expressing GFP gradually increased, and all the cells expressing GFP expressed RFP.

Three days after transfection, adherent cells were added to the bottom of the plate by adding trypsin and resuspended in PBS containing 2% FBS. Subsequently, the cells were suspended in single cells (single cell suspension), and the fluorescence labeled cell sorting was performed using a FACSAria II cell sorter (BD Biosciences, San Jose, CA, USA). To collect cells with mutations induced by nuclease, cells with RFP positive, GFP positive signals were sorted into 96-well plates. Cells transfected with only transfected cells and reporter were used as controls.

FIG. 2C shows, after 3 days of transfection, T84 cells transfected with a plasmid encoding a surrogate reporter plasmid and a CFTR guide RNA, and a plasmid encoding Cas9 and plasmid encoding Cas9, and T84 transfected with a plasmid encoding CFTR guide RNA In each cell, the degree of expression of RFP and GFP is represented by FACS. As shown in Fig. 2C, after 3 days of transfection, 3.2% of the cells showed RFP positive and GFP positive.

RFP positive and GFP positive cells were classified and the degree of mutation induced by Cas9 was measured by T7E1 assay. FIG. 2D shows the frequency of mutation in the target 2 region in exon 1 in a group of cells showing RFP-positive and GFP-positive by T7E1 analysis. As shown in FIG. 2D, the mutation frequency for the target 2 in T84 cells was about 39.7%, which proved that the CFTR gene was mutated by the guide RNA and the gene-modified cells were well obtained.

5. CFTR  Expression-inhibited clone selection

Each clone in the sorted cells was dispensed into 96-well plates. Among them, 7 kinds of clones were divided into 24 well plates and cultured. To determine whether genetic editing occurred in cultured cells, the exon 1 region was amplified using PCR from genomic DNA extracted from seven clonal populations.

Whether or not the mutation occurred in the target gene in the amplification product was detected by Sanger sequencing (Ran et al. 2013). The amplified product was purified and cloned into T-vector (T-vector) using T-Blunt PCR Cloning Kit (SolGent, Daejeon, South Korea). In each plate, 8 or 10 colonies were taken and cultured overnight, followed by plasmid mini-prep. The obtained DNA was sequenced using M13 (-20) reverse primer to confirm Cas9-induced NHEJ modification.

Figure 3A shows the DNA sequence of seven clones and the wild type T84 DNA sequence as a control group. The 20 bp target sequence complementary to the guide RNA was underlined. The 3 bp TAM sequence was blue. The deleted sequence was marked with -. The inserted sequence was indicated in red. The number of sequences deleted in parentheses () is indicated by -, and the number of inserted sequences is indicated by +. As shown in FIG. 3A, six clones (# 9, # 11, # 13, # 15, # 30, and # 34 clones) among the seven clones had mutations. On the other hand, of the seven clones, one clone (# 22 clone) did not have a mutation. Among the seven clones, the # 34 clone had a nucleotide deletion mutation of 17 bp on the homologous chromosome (allele) 1, a nucleotide insertion mutation of 4 bp, and a homologous chromosome (allele, allele) 2 to 15 bp nucleotide deletion mutation and 10 bp nucleotide insertion mutation. The mutation in clone # 34 caused a frame-shift mutation by insertion / deletion (indel) and generated a new start codon, resulting in a functionally inhibited CFTR gene product.

FIG. 3B is a Western blot graph showing the complete suppression of CFTR expression in # 34 clones in which insertion / deletion occurred, and the degree of CFTR expression in # 22 clones and wild type in which gene editing did not occur. HEK293T was transfected with a vector encoding ΔF508-CFTR and the ΔF508-CFTR protein over-expressed in HEK293T cells was used as a positive control. Aldolase expression was confirmed, and it was confirmed that the same amount of protein was used in Western blot analysis. As shown in Figure 3B, CFTR protein was hardly expressed in # 34 clone. On the other hand, CFTR protein was clearly expressed in wild-type T84 cells and in # 22 clone where genetic editing did not occur.

6. Virus generation

A lentiviral vector encoding ΔF508-CFTR was constructed as follows (Lee et al. 2015). To construct a virus encoding ΔF508-CFTR, the gene encoding ΔF508-CFTR was inserted into the Xbal restriction enzyme site of the lentiviral vector, pLenti6P. The gene encoding the? F508-CFTR comprises the nucleic acid sequence of SEQ ID NO: 16. Approximately 0.7 x 10 ⁶ HEK293T cells were plated in 6 well plates one day before transfection. The lentivirus plasmid pLenti6P-ΔF508-CFTR, the packaging vector psPAX2 and the envelope vector pMD2.G were mixed in a ratio of 3: 3: 1. Subsequently, HEK293T cells were transfected with 3 쨉 g of mixed DNA using opti-MEM (Invitrogen) containing lipofectamine 2000 for 6 hours. After transfection, the medium was replaced with 1.5 ml of DMEM / F-12 to remove the transfection reagent. One day after transfection, the medium containing lentiviral particles was collected, and the virus was obtained 24 hours later. The medium containing the virus particles was centrifuged at 3000 rpm for 5 minutes to remove the residue and stored at -80 캜.

7. Using a virus infection ΔF508 - CFTR  (Stable cell line), T84- ΔF508  making

A stable cell line expressing? F508-CFTR was constructed based on T84 cell # 34 clone using the lentiviral vector system. On the day prior to viral infection, T84 cell # 34 clones were dispensed into 12 well plates to approximately 10 ⁶ cells per well. The culture medium was exchanged with fresh medium containing polybrene at 8 占 퐂 / ml (Sigma) and cultured for 8 hours. The supernatant was then replaced with 0.5 ml of fresh medium containing 0.5 ml of lentiviral particles and incubated overnight. After 24 hours of transfection, the medium was replaced with fresh medium. Three days after virus infection, the medium was replaced with fresh medium containing 10 ug / ml of puromycin (Sigma). After two weeks, clones resistant to puromycin integrated with virus were selected and cultured. When the medium began to change to yellow, fresh medium was added. Colonies that apparently form clones were transferred to a 24 well plate and colonies were taken.

FIG. 3C shows the expression of ΔF508-CFTR in the prepared cell line by Western blotting. The T84 cell line was used as a control. HEK293T was transfected with a vector encoding ΔF508-CFTR and the ΔF508-CFTR protein over-expressed in HEK293T cells was used as a positive control. Aldolase expression was confirmed, and it was confirmed that the same amount of protein was used in Western blot analysis. As shown in Fig. 3C, ΔF508-CFTR was introduced into T84 cell # 34 clone, confirming that ΔF508-CFTR protein was expressed. The thus prepared stabilized cell line was named T84-ΔF508. The T84-ΔF508 cell line was deposited with the Korean Cell Line Bank under accession number KCLRF-BP-00388.

8. T84- ΔF508  Evaluation of the usefulness of the cell line

Whether or not the functional activity of ΔF508-CFTR was restored in T84-ΔF508 cells was monitored by the expression level of yellow fluorescent protein (YFP) sensitive to halide (Namkung et al., 2013). At this time, the principle that the halide anion permeates the cell membrane through CFTR was used.

T84-ΔF508 cells were transfected with halide-sensitive YFP (F46L / H148Q / I152L) (Dr. Alan Verkman, University of California, San Francisco). T84-ΔF508 cells were dispensed into 96 well black-walled microplates. After the cells were confluent to the plate bottom, the cells were incubated for 24 h at 27 ° C., which was a relatively low temperature, or for 24 h, with 10 μM of VX-809.

YFP quenching analysis was performed using a FLUO star Omega microplate reader (BMG Labtech, Ortenberg, Germany) and MARS data analysis software (BMG Labtech). Each well of the 96-well plate was washed three times with 200 쨉 l of PBS. 100 쨉 l of PBS was added to each well, and ΔF508-CFTR was activated with 10 μM forskolin. The 96-well plate was transferred to a pre-heated microplate reader at 37 ° C, and the degree of YFP fluorescence due to iodide entry into each well was continuously measured. Specifically, fluorescence was measured for 2 s (baseline) at 500 ms per point in each well followed by the addition of 140 mM NaI, 5 mM KCl, 1 mM MgCl ₂ , 1 mM CaCl ₂ , 10 D-glucose, and HEPES ) Was added to 2 seconds and the YFP fluorescence was recorded for 14 seconds to analyze the? F508-CFTR mediated iodide entry. The initial iodide entry rate was determined by nonlinear regression after injection of iodide, based on the slope at which fluorescence initially decreased.

Fig. 4A is a fluorescence image showing the expression of halide-sensitive YFP in T84-ΔF508 cells. 4B shows the degree of YFP expression when T84-ΔF508 cells were treated by treatment with CFTR _inh -172 at 37 ° C, 27 ° C, and 27 ° C, respectively. As shown in FIG. 4B, when the cells were cultured at 37 ° C, the YFP signal was not significantly changed, whereas when the cells were cultured at a relatively low temperature of 27 ° C, ΔF508-CFTR gradually recovered, YFP signal gradually decreased. When treated with CFTR _inh -172, the restored ΔF508-CFTR completely inhibited iodoate entry.

When cells expressing .DELTA.F508-CFTR are cultured at a temperature lower than 30.degree. C., cell membrane expression of .DELTA.F508-CFTR can be increased by inhibiting ERQCS (Endoplasmic reticulum (ER) quality control system) (Randell et al., J. Clin Invest., 99: 1432-1444, 1997). VX-809 is known to neutralize folding defects in the ΔF508-CFTR protein by acting on NBD1 as a corrector of ΔF508-CFTR (Van Goor et al. 2011). That is, VX-809 acts as a chaperone in protein folding and increases the number of CFTR proteins transported to the cell surface. VX-770, on the other hand, is a potentiator of CFTR, a substance that increases the open time of acinar CFTR in an ATP-dependent manner (Okiyoneda et al. 2013).

FIG. 4C shows the degree of YFP expression when T84-ΔF508 cells were treated with VX-809, VX-809, VX-770, and VX-809 and CFTR _inh -172, respectively. T84-ΔF508 cells were cultured with VX-809 for 24 hours to confirm that VX-809 restored ΔF508-CFTR function in T84-ΔF508 cells. Treatment of VX-809 significantly increased iodide entry through the recovered ΔF508-CFTR and increased iodide entry through the recovered ΔF508-CFTR when treated with VX-770. In addition, iodide entry induced by VX-809 was completely inhibited by CFTR _inh -172. Figure 4D shows the activity of the CFTR channel when treated with T84-ΔF508 cells treated at 37 ° C and 27 ° C respectively with VX-809, VX-809 and VX-770, and VX-809 with CFTR _inh -172 As shown in the graph. IUD508-CFTR-expressing CFTR knockout T84 cells were checked for iodide entry. As a result, iodide inflow was not observed. 4E shows Western blots as to whether band C was formed when T84-ΔF508 cells were reacted at 37 ° C., 27 ° C., and VX-809 treatment, respectively. Aldolase expression was confirmed, and it was confirmed that the same amount of protein was used in Western blot analysis. When T84-ΔF508 cells were cultured at a relatively low temperature of 27 ° C. and 10 μM of VX-809 for 24 hours, a band C of ΔF508 was clearly observed. From these results, it can be seen that the T84-ΔF508 cell line is a useful model for measuring the function of the recovered ΔF508-CFTR.

Depositor Name: Korea Cell Line Research Foundation

Accession number: KCLRFBP00388

Checked on: 20161115

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Guide RNA sequences for CFTR gene deletion, T84 cell having          mutated CFTR protein and use thereof <130> PN116634 <160> 17 <170> Kopatentin 2.0 <210> 1 <211> 6132 <212> DNA <213> Homo sapiens <400> 1 aattggaagc aaatgacatc acagcaggtc agagaaaaag ggttgagcgg caggcaccca 60 gagtagtagg tctttggcat taggagcttg agcccagacg gccctagcag ggaccccagc 120 gcccgagaga ccatgcagag gtcgcctctg gaaaaggcca gcgttgtctc caaacttttt 180 ttcagctgga ccagaccaat tttgaggaaa ggatacagac agcgcctgga attgtcagac 240 atataccaaa tcccttctgt tgattctgct gacaatctat ctgaaaaatt ggaaagagaa 300 tgggatagag agctggcttc aaagaaaaat cctaaactca ttaatgccct tcggcgatgt 360 tttttctgga gatttatgtt ctatggaatc tttttatatt taggggaagt caccaaagca 420 gtacagcctc tcttactggg aagaatcata gcttcctatg acccggataa caaggaggaa 480 cgctctatcg cgatttatct aggcataggc ttatgccttc tctttattgt gaggacactg 540 ctcctacacc cagccatttt tggccttcat cacattggaa tgcagatgag aatagctatg 600 tttagtttga tttataagaa gactttaaag ctgtcaagcc gtgttctaga taaaataagt 660 attggacaac ttgttagtct cctttccaac aacctgaaca aatttgatga aggacttgca 720 ttggcacatt tcgtgtggat cgctcctttg caagtggcac tcctcatggg gctaatctgg 780 gagttgttac aggcgtctgc cttctgtgga cttggtttcc tgatagtcct tgcccttttt 840 caggctgggc tagggagaat gatgatgaag tacagagatc agagagctgg gaagatcagt 900 gaaagacttg tgattacctc agaaatgatt gaaaatatcc aatctgttaa ggcatactgc 960 tgggaagaag caatggaaaa aatgattgaa aacttaagac aaacagaact gaaactgact 1020 cggaaggcag cctatgtgag atacttcaat agctcagcct tcttcttctc agggttcttt 1080 gtggtgtttt tatctgtgct tccctatgca ctaatcaaag gaatcatcct ccggaaaata 1140 ttcaccacca tctcattctg cattgttctg cgcatggcgg tcactcggca atttccctgg 1200 gctgtacaaa catggtatga ctctcttgga gcaataaaca aaatacagga tttcttacaa 1260 aagcaagaat ataagacatt ggaatataac ttaacgacta cagaagtagt gatggagaat 1320 gtaacagcct tctgggagga gggatttggg gaattatttg agaaagcaaa acaaaacaat 1380 aacaatagaa aaacttctaa tggtgatgac agcctcttct tcagtaattt ctcacttctt 1440 ggtactcctg tcctgaaaga tattaatttc aagatagaaa gaggacagtt gttggcggtt 1500 gctggatcca ctggagcagg caagacttca cttctaatgg tgattatggg agaactggag 1560 ccttcagagg gtaaaattaa gcacagtgga agaatttcat tctgttctca gttttcctgg 1620 attatgcctg gcaccattaa agaaaatatc atctttggtg tttcctatga tgaatataga 1680 tacagaagcg tcatcaaagc atgccaacta gaagaggaca tctccaagtt tgcagagaaa 1740 gacaatatag ttcttggaga aggtggaatc acactgagtg gaggtcaacg agcaagaatt 1800 tctttagcaa gagcagtata caaagatgct gatttgtatt tattagactc tccttttgga 1860 tacctagatg ttttaacaga aaaagaaata tttgaaagct gtgtctgtaa actgatggct 1920 aacaaaacta ggattttggt cacttctaaa atggaacatt taaagaaagc tgacaaaata 1980 ttaattttgc atgaaggtag cagctatttt tatgggacat tttcagaact ccaaaatcta 2040 cagccagact ttagctcaaa actcatggga tgtgattctt tcgaccaatt tagtgcagaa 2100 agaagaaatt caatcctaac tgagacctta caccgtttct cattagaagg agatgctcct 2160 gtctcctgga cagaaacaaa aaaacaatct tttaaacaga ctggagagtt tggggaaaaa 2220 aggaagaatt ctattctcaa tccaatcaac tctatacgaa aattttccat tgtgcaaaag 2280 actcccttac aaatgaatgg catcgaagag gattctgatg agcctttaga gagaaggctg 2340 tccttagtac cagattctga gcagggagag gcgatactgc ctcgcatcag cgtgatcagc 2400 actggcccca cgcttcaggc acgaaggagg cagtctgtcc tgaacctgat gacacactca 2460 gttaaccaag gtcagaacat tcaccgaaag acaacagcat ccacacgaaa agtgtcactg 2520 gcccctcagg caaacttgac tgaactggat atatattcaa gaaggttatc tcaagaaact 2580 ggcttggaaa taagtgaaga aattaacgaa gaagacttaa aggagtgctt ttttgatgat 2640 atggagagca taccagcagt gactacatgg aacacatacc ttcgatatat tactgtccac 2700 aagagcttaa tttttgtgct aatttggtgc ttagtaattt ttctggcaga ggtggctgct 2760 tctttggttg tgctgtggct ccttggaaac actcctcttc aagacaaagg gaatagtact 2820 catagtagaa ataacagcta tgcagtgatt atcaccagca ccagttcgta ttatgtgttt 2880 tacatttacg tgggagtagc cgacactttg cttgctatgg gattcttcag aggtctacca 2940 ctggtgcata ctctaatcac agtgtcgaaa attttacacc acaaaatgtt acattctgtt 3000 cttcaagcac ctatgtcaac cctcaacacg ttgaaagcag gtgggattct taatagattc 3060 tccaaagata tagcaatttt ggatgacctt ctgcctctta ccatatttga cttcatccag 3120 ttgttattaa ttgtgattgg agctatagca gttgtcgcag ttttacaacc ctacatcttt 3180 gttgcaacag tgccagtgat agtggctttt attatgttga gagcatattt cctccaaacc 3240 tcacagcaac tcaaacaact ggaatctgaa ggcaggagtc caattttcac tcatcttgtt 3300 acaagcttaa aaggactatg gacacttcgt gccttcggac ggcagcctta ctttgaaact 3360 ctgttccaca aagctctgaa tttacatact gccaactggt tcttgtacct gtcaacactg 3420 cgctggttcc aaatgagaat agaaatgatt tttgtcatct tcttcattgc tgttaccttc 3480 atttccattt taacaacagg agaaggagaa ggaagagttg gtattatcct gactttagcc 3540 atgaatatca tgagtacatt gcagtgggct gtaaactcca gcatagatgt ggatagcttg 3600 atgcgatctg tgagccgagt ctttaagttc attgacatgc caacagaagg taaacctacc 3660 aagtcaacca aaccatacaa gaatggccaa ctctcgaaag ttatgattat tgagaattca 3720 cacgtgaaga aagatgacat ctggccctca gggggccaaa tgactgtcaa agatctcaca 3780 gcaaaataca cagaaggtgg aaatgccata ttagagaaca tttccttctc aataagtcct 3840 ggccagaggg tgggcctctt gggaagaact ggatcaggga agagtacttt gttatcagct 3900 tttttgagac tactgaacac tgaaggagaa atccagatcg atggtgtgtc ttgggattca 3960 ataactttgc aacagtggag gaaagccttt ggagtgatac cacagaaagt atttattttt 4020 tctggaacat ttagaaaaaa cttggatccc tatgaacagt ggagtgatca agaaatatgg 4080 aaagttgcag atgaggttgg gctcagatct gtgatagaac agtttcctgg gaagcttgac 4140 tttgtccttg tggatggggg ctgtgtccta agccatggcc acaagcagtt gatgtgcttg 4200 gctagatctg ttctcagtaa ggcgaagatc ttgctgcttg atgaacccag tgctcatttg 4260 gatccagtaa cataccaaat aattagaaga actctaaaac aagcatttgc tgattgcaca 4320 gtaattctct gtgaacacag gatagaagca atgctggaat gccaacaatt tttggtcata 4380 gaagagaaca aagtgcggca gtacgattcc atccagaaac tgctgaacga gaggagcctc 4440 ttccggcaag ccatcagccc ctccgacagg gtgaagctct ttccccaccg gaactcaagc 4500 aagtgcaagt ctaagcccca gattgctgct ctgaaagagg agacagaaga agaggtgcaa 4560 gatacaaggc tttagagagc agcataaatg ttgacatggg acatttgctc atggaattgg 4620 agctcgtggg acagtcacct catggaattg gagctcgtgg aacagttacc tctgcctcag 4680 aaaacaagga tgaattaagt ttttttttaa aaaagaaaca tttggtaagg ggaattgagg 4740 acactgatat gggtcttgat aaatggcttc ctggcaatag tcaaattgtg tgaaaggtac 4800 ttcaaatcct tgaagattta ccacttgtgt tttgcaagcc agattttcct gaaaaccctt 4860 gccatgtgct agtaattgga aaggcagctc taaatgtcaa tcagcctagt tgatcagctt 4920 attgtctagt gaaactcgtt aatttgtagt gttggagaag aactgaaatc atacttctta 4980 gggttatgat taagtaatga taactggaaa cttcagcggt ttatataagc ttgtattcct 5040 ttttctctcc tctccccatg atgtttagaa acacaactat attgtttgct aagcattcca 5100 actatctcat ttccaagcaa gtattagaat accacaggaa ccacaagact gcacatcaaa 5160 atatgcccca ttcaacatct agtgagcagt caggaaagag aacttccaga tcctggaaat 5220 cagggttagt attgtccagg tctaccaaaa atctcaatat ttcagataat cacaatacat 5280 cccttacctg ggaaagggct gttataatct ttcacagggg acaggatggt tcccttgatg 5340 aagaagttga tatgcctttt cccaactcca gaaagtgaca agctcacaga cctttgaact 5400 agagtttagc tggaaaagta tgttagtgca aattgtcaca ggacagccct tctttccaca 5460 gaagctccag gtagagggtg tgtaagtaga taggccatgg gcactgtggg tagacacaca 5520 tgaagtccaa gcatttagat gtataggttg atggtggtat gttttcaggc tagatgtatg 5580 tacttcatgc tgtctacact aagagagaat gagagacaca ctgaagaagc accaatcatg 5640 aattagtttt atatgcttct gttttataat tttgtgaagc aaaatttttt ctctaggaaa 5700 tatttatttt aataatgttt caaacatata taacaatgct gtattttaaa agaatgatta 5760 tgaattacat ttgtataaaa taatttttat atttgaaata ttgacttttt atggcactag 5820 tatttctatg aaatattatg ttaaaactgg gacaggggag aacctagggt gatattaacc 5880 aggggccatg aatcaccttt tggtctggag ggaagccttg gggctgatgc agttgttgcc 5940 cacagctgta tgattcccag ccagcacagc ctcttagatg cagttctgaa gaagatggta 6000 ccaccagtct gactgtttcc atcaagggta cactgccttc tcaactccaa actgactctt 6060 aagaagactg cattatattt attactgtaa gaaaatatca cttgtcaata aaatccatac 6120 atttgtgtga aa 6132 <210> 2 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> guide RNA for target1 <400> 2 gaggcgaccu cugcaugguc 20 <210> 3 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> guide RNA for target2 <400> 3 cugcaugguc ucucgggcgc 20 <210> 4 <211> 20 <212> RNA <213> Artificial Sequence <220> <223> guide RNA for target3 <400> 4 caaaauuggu cugguccagc 20 <210> 5 <211> 1480 <212> PRT <213> Homo sapiens <400> 5 Met Gln Arg Ser Ser Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe   1 5 10 15 Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gly Tyr Arg Gln Arg Leu              20 25 30 Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Val Asp Ser Ala Asp Asn          35 40 45 Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu Leu Ala Ser Lys      50 55 60 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg  65 70 75 80 Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gly Glu Val Thr Lys Ala                  85 90 95 Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp             100 105 110 Asn Lys Glu Glu Arg Ser Ile Ale Ile Tyr Leu Gly Ile Gly Leu Cys         115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly     130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser                 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp             180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val         195 200 205 Ala Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe     210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser                 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val             260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu         275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr     290 295 300 Phe Asn Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile                 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg             340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile         355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu     370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Gly Phe Gly Glu Leu Phe Glu Lys Ala Lys Gln Asn Asn                 405 410 415 Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Ser Leu Phe Phe Ser Asn             420 425 430 Phe Ser Leu Leu Gly Thr Pro Val Leu Lys Asp Ile Asn Phe Lys Ile         435 440 445 Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Ser Thr Gly Ala Gly Lys     450 455 460 Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro Ser Glu Gly 465 470 475 480 Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln Phe Ser Trp                 485 490 495 Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Phe Gly Val Ser Tyr             500 505 510 Asp Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln Leu Glu Glu         515 520 525 Asp Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu Gly Glu Gly     530 535 540 Gly Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser Leu Ala Arg 545 550 555 560 Ala Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser Pro Phe Gly                 565 570 575 Tyr Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser Cys Val Cys             580 585 590 Lys Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser Lys Met Glu         595 600 605 His Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu Gly Ser Ser     610 615 620 Tyr Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln Pro Asp Phe 625 630 635 640 Ser Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe Ser Ala Glu                 645 650 655 Arg Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe Ser Leu Glu             660 665 670 Gly Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln Ser Phe Lys         675 680 685 Gln Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro     690 695 700 Ile Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr Pro Leu Gln 705 710 715 720 Met Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu Arg Arg Leu                 725 730 735 Ser Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu Pro Arg Ile             740 745 750 Ser Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg Arg Gln Ser         755 760 765 Val Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln Asn Ile His     770 775 780 Arg Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gln Ala 785 790 795 800 Asn Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gln Glu Thr                 805 810 815 Gly Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys             820 825 830 Phe Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr Trp Asn Thr         835 840 845 Tyr Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile     850 855 860 Trp Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser Leu Val Val 865 870 875 880 Leu Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly Asn Ser Thr                 885 890 895 His Ser Arg Asn As Ser Tyr Ala Val Ile Ile Thr Ser Thr Ser Ser             900 905 910 Tyr Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr Leu Leu Ala         915 920 925 Met Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val     930 935 940 Ser Lys Ile Leu His His Lys Met Leu His Ser Val Leu Gln Ala Pro 945 950 955 960 Met Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe                 965 970 975 Ser Lys Asp Ile Ala Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe             980 985 990 Asp Phe Ile Gln Leu Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val         995 1000 1005 Ala Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile Val    1010 1015 1020 Ala Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser Gln Gln Leu 1025 1030 1035 1040 Lys Gln Leu Glu Ser Glu Gly Arg Ser Ser Ile Phe Thr His Leu Val                1045 1050 1055 Thr Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe Gly Arg Gln Pro            1060 1065 1070 Tyr Phe Glu Thr Leu Phe His Lys Ala Leu Asn Leu His Thr Ala Asn        1075 1080 1085 Trp Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe Gln Met Arg Ile Glu    1090 1095 1100 Met Ile Phe Val Ile Phe Phe Ile Ala Val Thr Phe Ile Ser Ile Leu 1105 1110 1115 1120 Thr Thr Gly Glu Gly Glu Gly Arg Val Gly Ile Ile Leu Thr Leu Ala                1125 1130 1135 Met Asn Ile Met Ser Thr Leu Gln Trp Ala Val Asn Ser Ser Ile Asp            1140 1145 1150 Val Asp Ser Leu Met Arg Ser Val Ser Arg Val Phe Lys Phe Ile Asp        1155 1160 1165 Met Pro Thr Glu Gly Lys Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn    1170 1175 1180 Gly Gln Leu Ser Lys Val Met Ile Ile Glu Asn Ser His Val Lys Lys 1185 1190 1195 1200 Asp Asp Ile Trp Pro Ser Gly Gly Gln Met Thr Val Lys Asp Leu Thr                1205 1210 1215 Ala Lys Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe            1220 1225 1230 Ser Ile Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg Thr Gly Ser        1235 1240 1245 Gly Lys Ser Thr Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr Glu    1250 1255 1260 Gly Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile Thr Leu Gln 1265 1270 1275 1280 Gln Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val Phe Ile Phe                1285 1290 1295 Ser Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu Gln Trp Ser Asp            1300 1305 1310 Gln Glu Ile Trp Lys Val Ala Asp Glu Val Gly Leu Arg Ser Val Ile        1315 1320 1325 Glu Gln Phe Pro Gly Lys Leu Asp Phe Val Leu Val Asp Gly Gly Cys    1330 1335 1340 Val Leu Ser His Gly His Lys Gln Leu Met Cys Leu Ala Arg Ser Val 1345 1350 1355 1360 Leu Ser Lys Ala Lys Ile Leu Leu Leu Asp Glu Pro Ser Ala His Leu                1365 1370 1375 Asp Pro Val Thr Tyr Gln Ile Ile Arg Arg Thr Leu Lys Gln Ala Phe            1380 1385 1390 Ala Asp Cys Thr Val Ile Leu Cys Glu His Arg Ile Glu Ala Met Leu        1395 1400 1405 Glu Cys Gln Gln Phe Leu Val Ile Glu Glu Asn Lys Val Arg Gln Tyr    1410 1415 1420 Asp Ser Ile Gln Lys Leu Leu Asn Glu Arg Ser Leu Phe Arg Gln Ala 1425 1430 1435 1440 Ile Ser Pro Ser Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser                1445 1450 1455 Lys Cys Lys Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu Thr Glu            1460 1465 1470 Glu Glu Val Gln Asp Thr Arg Leu        1475 1480 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target1 <400> 6 gaccatgcag aggtcgcctc 20 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target1 <400> 7 gaggcgacct ctgcatggtc 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target2 <400> 8 gcgcccgaga gaccatgcag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target2 <400> 9 ctgcatggtc tctcgggcgc 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target3 <400> 10 gctggaccag accaattttg 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> complementary oligos for target3 <400> 11 caaaattggt ctggtccagc 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> F-primer for exon1 <400> 12 aattggaagc aaatgacatc 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> R-primer for exon1 <400> 13 tgaagaatca tttacctgtg 20 <210> 14 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> F-primer for exon2 <400> 14 gaggctggac ttcagtaatt tg 22 <210> 15 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> R-primer for exon2 <400> 15 caagtaaccc tcctgcttca g 21 <210> 16 <211> 6129 <212> DNA <213> Homo sapiens <400> 16 aattggaagc aaatgacatc acagcaggtc agagaaaaag ggttgagcgg caggcaccca 60 gagtagtagg tctttggcat taggagcttg agcccagacg gccctagcag ggaccccagc 120 gcccgagaga ccatgcagag gtcgcctctg gaaaaggcca gcgttgtctc caaacttttt 180 ttcagctgga ccagaccaat tttgaggaaa ggatacagac agcgcctgga attgtcagac 240 atataccaaa tcccttctgt tgattctgct gacaatctat ctgaaaaatt ggaaagagaa 300 tgggatagag agctggcttc aaagaaaaat cctaaactca ttaatgccct tcggcgatgt 360 tttttctgga gatttatgtt ctatggaatc tttttatatt taggggaagt caccaaagca 420 gtacagcctc tcttactggg aagaatcata gcttcctatg acccggataa caaggaggaa 480 cgctctatcg cgatttatct aggcataggc ttatgccttc tctttattgt gaggacactg 540 ctcctacacc cagccatttt tggccttcat cacattggaa tgcagatgag aatagctatg 600 tttagtttga tttataagaa gactttaaag ctgtcaagcc gtgttctaga taaaataagt 660 attggacaac ttgttagtct cctttccaac aacctgaaca aatttgatga aggacttgca 720 ttggcacatt tcgtgtggat cgctcctttg caagtggcac tcctcatggg gctaatctgg 780 gagttgttac aggcgtctgc cttctgtgga cttggtttcc tgatagtcct tgcccttttt 840 caggctgggc tagggagaat gatgatgaag tacagagatc agagagctgg gaagatcagt 900 gaaagacttg tgattacctc agaaatgatt gaaaatatcc aatctgttaa ggcatactgc 960 tgggaagaag caatggaaaa aatgattgaa aacttaagac aaacagaact gaaactgact 1020 cggaaggcag cctatgtgag atacttcaat agctcagcct tcttcttctc agggttcttt 1080 gtggtgtttt tatctgtgct tccctatgca ctaatcaaag gaatcatcct ccggaaaata 1140 ttcaccacca tctcattctg cattgttctg cgcatggcgg tcactcggca atttccctgg 1200 gctgtacaaa catggtatga ctctcttgga gcaataaaca aaatacagga tttcttacaa 1260 aagcaagaat ataagacatt ggaatataac ttaacgacta cagaagtagt gatggagaat 1320 gtaacagcct tctgggagga gggatttggg gaattatttg agaaagcaaa acaaaacaat 1380 aacaatagaa aaacttctaa tggtgatgac agcctcttct tcagtaattt ctcacttctt 1440 ggtactcctg tcctgaaaga tattaatttc aagatagaaa gaggacagtt gttggcggtt 1500 gctggatcca ctggagcagg caagacttca cttctaatgg tgattatggg agaactggag 1560 ccttcagagg gtaaaattaa gcacagtgga agaatttcat tctgttctca gttttcctgg 1620 attatgcctg gcaccattaa agaaaatatc attggtgttt cctatgatga atatagatac 1680 agaagcgtca tcaaagcatg ccaactagaa gaggacatct ccaagtttgc agagaaagac 1740 aatatagttc ttggagaagg tggaatcaca ctgagtggag gtcaacgagc aagaatttct 1800 ttagcaagag cagtatacaa agatgctgat ttgtatttat tagactctcc ttttggatac 1860 ctagatgttt taacagaaaa agaaatattt gaaagctgtg tctgtaaact gatggctaac 1920 aaaactagga ttttggtcac ttctaaaatg gaacatttaa agaaagctga caaaatatta 1980 attttgcatg aaggtagcag ctatttttat gggacatttt cagaactcca aaatctacag 2040 ccagacttta gctcaaaact catgggatgt gattctttcg accaatttag tgcagaaaga 2100 agaaattcaa tcctaactga gaccttacac cgtttctcat tagaaggaga tgctcctgtc 2160 tcctggacag aaacaaaaaa acaatctttt aaacagactg gagagtttgg ggaaaaaagg 2220 aagaattcta ttctcaatcc aatcaactct atacgaaaat tttccattgt gcaaaagact 2280 cccttacaaa tgaatggcat cgaagaggat tctgatgagc ctttagagag aaggctgtcc 2340 ttagtaccag attctgagca gggagaggcg atactgcctc gcatcagcgt gatcagcact 2400 ggccccacgc ttcaggcacg aaggaggcag tctgtcctga acctgatgac acactcagtt 2460 aaccaaggtc agaacattca ccgaaagaca acagcatcca cacgaaaagt gtcactggcc 2520 cctcaggcaa acttgactga actggatata tattcaagaa ggttatctca agaaactggc 2580 ttggaaataa gtgaagaaat taacgaagaa gacttaaagg agtgcttttt tgatgatatg 2640 gagagcatac cagcagtgac tacatggaac acataccttc gatatattac tgtccacaag 2700 agcttaattt ttgtgctaat ttggtgctta gtaatttttc tggcagaggt ggctgcttct 2760 ttggttgtgc tgtggctcct tggaaacact cctcttcaag acaaagggaa tagtactcat 2820 agtagaaata acagctatgc agtgattatc accagcacca gttcgtatta tgtgttttac 2880 atttacgtgg gagtagccga cactttgctt gctatgggat tcttcagagg tctaccactg 2940 gtgcatactc taatcacagt gtcgaaaatt ttacaccaca aaatgttaca ttctgttctt 3000 caagcaccta tgtcaaccct caacacgttg aaagcaggtg ggattcttaa tagattctcc 3060 aaagatatag caattttgga tgaccttctg cctcttacca tatttgactt catccagttg 3120 ttattaattg tgattggagc tatagcagtt gtcgcagttt tacaacccta catctttgtt 3180 gcaacagtgc cagtgatagt ggcttttatt atgttgagag catatttcct ccaaacctca 3240 cagcaactca aacaactgga atctgaaggc aggagtccaa ttttcactca tcttgttaca 3300 agcttaaaag gactatggac acttcgtgcc ttcggacggc agccttactt tgaaactctg 3360 ttccacaaag ctctgaattt acatactgcc aactggttct tgtacctgtc aacactgcgc 3420 tggttccaaa tgagaataga aatgattttt gtcatcttct tcattgctgt taccttcatt 3480 tccattttaa caacaggaga aggagaagga agagttggta ttatcctgac tttagccatg 3540 aatatcatga gtacattgca gtgggctgta aactccagca tagatgtgga tagcttgatg 3600 cgatctgtga gccgagtctt taagttcatt gacatgccaa cagaaggtaa acctaccaag 3660 tcaaccaaac catacaagaa tggccaactc tcgaaagtta tgattattga gaattcacac 3720 gtgaagaaag atgacatctg gccctcaggg ggccaaatga ctgtcaaaga tctcacagca 3780 aaatacacag aaggtggaaa tgccatatta gagaacattt ccttctcaat aagtcctggc 3840 cagagggtgg gcctcttggg aagaactgga tcagggaaga gtactttgtt atcagctttt 3900 ttgagactac tgaacactga aggagaaatc cagatcgatg gtgtgtcttg ggattcaata 3960 actttgcaac agtggaggaa agcctttgga gtgataccac agaaagtatt tattttttct 4020 ggaacattta gaaaaaactt ggatccctat gaacagtgga gtgatcaaga aatatggaaa 4080 gttgcagatg aggttgggct cagatctgtg atagaacagt ttcctgggaa gcttgacttt 4140 gtccttgtgg atgggggctg tgtcctaagc catggccaca agcagttgat gtgcttggct 4200 agatctgttc tcagtaaggc gaagatcttg ctgcttgatg aacccagtgc tcatttggat 4260 ccagtaacat accaaataat tagaagaact ctaaaacaag catttgctga ttgcacagta 4320 attctctgtg aacacaggat agaagcaatg ctggaatgcc aacaattttt ggtcatagaa 4380 gagaacaaag tgcggcagta cgattccatc cagaaactgc tgaacgagag gagcctcttc 4440 cggcaagcca tcagcccctc cgacagggtg aagctctttc cccaccggaa ctcaagcaag 4500 tgcaagtcta agccccagat tgctgctctg aaagaggaga cagaagaaga ggtgcaagat 4560 acaaggcttt agagagcagc ataaatgttg acatgggaca tttgctcatg gaattggagc 4620 tcgtgggaca gtcacctcat ggaattggag ctcgtggaac agttacctct gcctcagaaa 4680 acaaggatga attaagtttt tttttaaaaa agaaacattt ggtaagggga attgaggaca 4740 ctgatatggg tcttgataaa tggcttcctg gcaatagtca aattgtgtga aaggtacttc 4800 aaatccttga agatttacca cttgtgtttt gcaagccaga ttttcctgaa aacccttgcc 4860 atgtgctagt aattggaaag gcagctctaa atgtcaatca gcctagttga tcagcttatt 4920 gtctagtgaa actcgttaat ttgtagtgtt ggagaagaac tgaaatcata cttcttaggg 4980 ttatgattaa gtaatgataa ctggaaactt cagcggttta tataagcttg tattcctttt 5040 tctctcctct ccccatgatg tttagaaaca caactatatt gtttgctaag cattccaact 5100 atctcatttc caagcaagta ttagaatacc acaggaacca caagactgca catcaaaata 5160 tgccccattc aacatctagt gagcagtcag gaaagagaac ttccagatcc tggaaatcag 5220 ggttagtatt gtccaggtct accaaaaatc tcaatatttc agataatcac aatacatccc 5280 ttacctggga aagggctgtt ataatctttc acaggggaca ggatggttcc cttgatgaag 5340 aagttgatat gccttttccc aactccagaa agtgacaagc tcacagacct ttgaactaga 5400 gtttagctgg aaaagtatgt tagtgcaaat tgtcacagga cagcccttct ttccacagaa 5460 gctccaggta gagggtgtgt aagtagatag gccatgggca ctgtgggtag acacacatga 5520 agtccaagca tttagatgta taggttgatg gtggtatgtt ttcaggctag atgtatgtac 5580 ttcatgctgt ctacactaag agagaatgag agacacactg aagaagcacc aatcatgaat 5640 tagttttata tgcttctgtt ttataatttt gtgaagcaaa attttttctc taggaaatat 5700 ttattttaat aatgtttcaa acatatataa caatgctgta ttttaaaaga atgattatga 5760 attacatttg tataaaataa tttttatatt tgaaatattg actttttatg gcactagtat 5820 ttctatgaaa tattatgtta aaactgggac aggggagaac ctagggtgat attaaccagg 5880 ggccatgaat caccttttgg tctggaggga agccttgggg ctgatgcagt tgttgcccac 5940 agctgtatga ttcccagcca gcacagcctc ttagatgcag ttctgaagaa gatggtacca 6000 ccagtctgac tgtttccatc aagggtacac tgccttctca actccaaact gactcttaag 6060 aagactgcat tatatttatt actgtaagaa aatatcactt gtcaataaaa tccatacatt 6120 tgtgtgaaa 6129 <210> 17 <211> 1479 <212> PRT <213> Homo sapiens <400> 17 Met Gln Arg Ser Ser Leu Glu Lys Ala Ser Val Val Ser Lys Leu Phe   1 5 10 15 Phe Ser Trp Thr Arg Pro Ile Leu Arg Lys Gly Tyr Arg Gln Arg Leu              20 25 30 Glu Leu Ser Asp Ile Tyr Gln Ile Pro Ser Val Asp Ser Ala Asp Asn          35 40 45 Leu Ser Glu Lys Leu Glu Arg Glu Trp Asp Arg Glu Leu Ala Ser Lys      50 55 60 Lys Asn Pro Lys Leu Ile Asn Ala Leu Arg Arg Cys Phe Phe Trp Arg  65 70 75 80 Phe Met Phe Tyr Gly Ile Phe Leu Tyr Leu Gly Glu Val Thr Lys Ala                  85 90 95 Val Gln Pro Leu Leu Leu Gly Arg Ile Ile Ala Ser Tyr Asp Pro Asp             100 105 110 Asn Lys Glu Glu Arg Ser Ile Ale Ile Tyr Leu Gly Ile Gly Leu Cys         115 120 125 Leu Leu Phe Ile Val Arg Thr Leu Leu Leu His Pro Ala Ile Phe Gly     130 135 140 Leu His His Ile Gly Met Gln Met Arg Ile Ala Met Phe Ser Leu Ile 145 150 155 160 Tyr Lys Lys Thr Leu Lys Leu Ser Ser Arg Val Leu Asp Lys Ile Ser                 165 170 175 Ile Gly Gln Leu Val Ser Leu Leu Ser Asn Asn Leu Asn Lys Phe Asp             180 185 190 Glu Gly Leu Ala Leu Ala His Phe Val Trp Ile Ala Pro Leu Gln Val         195 200 205 Ala Leu Met Gly Leu Ile Trp Glu Leu Leu Gln Ala Ser Ala Phe     210 215 220 Cys Gly Leu Gly Phe Leu Ile Val Leu Ala Leu Phe Gln Ala Gly Leu 225 230 235 240 Gly Arg Met Met Met Lys Tyr Arg Asp Gln Arg Ala Gly Lys Ile Ser                 245 250 255 Glu Arg Leu Val Ile Thr Ser Glu Met Ile Glu Asn Ile Gln Ser Val             260 265 270 Lys Ala Tyr Cys Trp Glu Glu Ala Met Glu Lys Met Ile Glu Asn Leu         275 280 285 Arg Gln Thr Glu Leu Lys Leu Thr Arg Lys Ala Ala Tyr Val Arg Tyr     290 295 300 Phe Asn Ser Ala Phe Phe Phe Ser Gly Phe Phe Val Val Phe Leu 305 310 315 320 Ser Val Leu Pro Tyr Ala Leu Ile Lys Gly Ile Ile Leu Arg Lys Ile                 325 330 335 Phe Thr Thr Ile Ser Phe Cys Ile Val Leu Arg Met Ala Val Thr Arg             340 345 350 Gln Phe Pro Trp Ala Val Gln Thr Trp Tyr Asp Ser Leu Gly Ala Ile         355 360 365 Asn Lys Ile Gln Asp Phe Leu Gln Lys Gln Glu Tyr Lys Thr Leu Glu     370 375 380 Tyr Asn Leu Thr Thr Thr Glu Val Val Met Glu Asn Val Thr Ala Phe 385 390 395 400 Trp Glu Gly Phe Gly Glu Leu Phe Glu Lys Ala Lys Gln Asn Asn                 405 410 415 Asn Asn Arg Lys Thr Ser Asn Gly Asp Asp Ser Leu Phe Phe Ser Asn             420 425 430 Phe Ser Leu Leu Gly Thr Pro Val Leu Lys Asp Ile Asn Phe Lys Ile         435 440 445 Glu Arg Gly Gln Leu Leu Ala Val Ala Gly Ser Thr Gly Ala Gly Lys     450 455 460 Thr Ser Leu Leu Met Val Ile Met Gly Glu Leu Glu Pro Ser Glu Gly 465 470 475 480 Lys Ile Lys His Ser Gly Arg Ile Ser Phe Cys Ser Gln Phe Ser Trp                 485 490 495 Ile Met Pro Gly Thr Ile Lys Glu Asn Ile Ile Gly Val Ser Tyr Asp             500 505 510 Glu Tyr Arg Tyr Arg Ser Val Ile Lys Ala Cys Gln Leu Glu Glu Asp         515 520 525 Ile Ser Lys Phe Ala Glu Lys Asp Asn Ile Val Leu Gly Glu Gly Gly Gly     530 535 540 Ile Thr Leu Ser Gly Gly Gln Arg Ala Arg Ile Ser Leu Ala Arg Ala 545 550 555 560 Val Tyr Lys Asp Ala Asp Leu Tyr Leu Leu Asp Ser Pro Phe Gly Tyr                 565 570 575 Leu Asp Val Leu Thr Glu Lys Glu Ile Phe Glu Ser Cys Val Cys Lys             580 585 590 Leu Met Ala Asn Lys Thr Arg Ile Leu Val Thr Ser Lys Met Glu His         595 600 605 Leu Lys Lys Ala Asp Lys Ile Leu Ile Leu His Glu Gly Ser Ser Tyr     610 615 620 Phe Tyr Gly Thr Phe Ser Glu Leu Gln Asn Leu Gln Pro Asp Phe Ser 625 630 635 640 Ser Lys Leu Met Gly Cys Asp Ser Phe Asp Gln Phe Ser Ala Glu Arg                 645 650 655 Arg Asn Ser Ile Leu Thr Glu Thr Leu His Arg Phe Ser Leu Glu Gly             660 665 670 Asp Ala Pro Val Ser Trp Thr Glu Thr Lys Lys Gln Ser Phe Lys Gln         675 680 685 Thr Gly Glu Phe Gly Glu Lys Arg Lys Asn Ser Ile Leu Asn Pro Ile     690 695 700 Asn Ser Ile Arg Lys Phe Ser Ile Val Gln Lys Thr Pro Leu Gln Met 705 710 715 720 Asn Gly Ile Glu Glu Asp Ser Asp Glu Pro Leu Glu Arg Arg Leu Ser                 725 730 735 Leu Val Pro Asp Ser Glu Gln Gly Glu Ala Ile Leu Pro Arg Ile Ser             740 745 750 Val Ile Ser Thr Gly Pro Thr Leu Gln Ala Arg Arg Arg Gln Ser Val         755 760 765 Leu Asn Leu Met Thr His Ser Val Asn Gln Gly Gln Asn Ile His Arg     770 775 780 Lys Thr Thr Ala Ser Thr Arg Lys Val Ser Leu Ala Pro Gln Ala Asn 785 790 795 800 Leu Thr Glu Leu Asp Ile Tyr Ser Arg Arg Leu Ser Gln Glu Thr Gly                 805 810 815 Leu Glu Ile Ser Glu Glu Ile Asn Glu Glu Asp Leu Lys Glu Cys Phe             820 825 830 Phe Asp Asp Met Glu Ser Ile Pro Ala Val Thr Thr Trp Asn Thr Tyr         835 840 845 Leu Arg Tyr Ile Thr Val His Lys Ser Leu Ile Phe Val Leu Ile Trp     850 855 860 Cys Leu Val Ile Phe Leu Ala Glu Val Ala Ala Ser Leu Val Val Leu 865 870 875 880 Trp Leu Leu Gly Asn Thr Pro Leu Gln Asp Lys Gly Asn Ser Thr His                 885 890 895 Ser Arg Asn Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser Ser             900 905 910 Tyr Val Phe Tyr Ile Tyr Val Gly Val Ala Asp Thr Leu Leu Ala Met         915 920 925 Gly Phe Phe Arg Gly Leu Pro Leu Val His Thr Leu Ile Thr Val Ser     930 935 940 Lys Ile Leu His His Lys Met Leu His Ser Val Leu Gln Ala Pro Met 945 950 955 960 Ser Thr Leu Asn Thr Leu Lys Ala Gly Gly Ile Leu Asn Arg Phe Ser                 965 970 975 Lys Asp Ile Ale Ile Leu Asp Asp Leu Leu Pro Leu Thr Ile Phe Asp             980 985 990 Phe Ile Gln Leu Leu Ile Val Ile Gly Ala Ile Ala Val Val Ala         995 1000 1005 Val Leu Gln Pro Tyr Ile Phe Val Ala Thr Val Pro Val Ile Val Ala    1010 1015 1020 Phe Ile Met Leu Arg Ala Tyr Phe Leu Gln Thr Ser Gln Gln Leu Lys 1025 1030 1035 1040 Gln Leu Glu Ser Glu Gly Arg Ser Pro Ile Phe Thr His Leu Val Thr                1045 1050 1055 Ser Leu Lys Gly Leu Trp Thr Leu Arg Ala Phe Gly Arg Gln Pro Tyr            1060 1065 1070 Phe Glu Thr Leu Phe His Lys Ala Leu Asn Leu His Thr Ala Asn Trp        1075 1080 1085 Phe Leu Tyr Leu Ser Thr Leu Arg Trp Phe Gln Met Arg Ile Glu Met    1090 1095 1100 Ile Phe Val Ile Phe Phe Ile Ala Val Thr Phe Ile Ser Ile Leu Thr 1105 1110 1115 1120 Thr Gly Glu Gly Glu Gly Arg Val Gly Ile Ile Leu Thr Leu Ala Met                1125 1130 1135 Asn Ile Met Ser Thr Leu Gln Trp Ala Val Asn Ser Ser Ile Asp Val            1140 1145 1150 Asp Ser Leu Met Arg Ser Val Ser Ser Val Val Phe Lys Phe Ile Asp Met        1155 1160 1165 Pro Thr Glu Gly Lys Pro Thr Lys Ser Thr Lys Pro Tyr Lys Asn Gly    1170 1175 1180 Gln Leu Ser Lys Val Met Ile Ile Glu Asn Ser His Val Lys Lys Asp 1185 1190 1195 1200 Asp Ile Trp Pro Ser Gly Gly Gln Met Thr Val Lys Asp Leu Thr Ala                1205 1210 1215 Lys Tyr Thr Glu Gly Gly Asn Ala Ile Leu Glu Asn Ile Ser Phe Ser            1220 1225 1230 Ile Ser Pro Gly Gln Arg Val Gly Leu Leu Gly Arg Thr Gly Ser Gly        1235 1240 1245 Lys Ser Thr Leu Leu Ser Ala Phe Leu Arg Leu Leu Asn Thr Glu Gly    1250 1255 1260 Glu Ile Gln Ile Asp Gly Val Ser Trp Asp Ser Ile Thr Leu Gln Gln 1265 1270 1275 1280 Trp Arg Lys Ala Phe Gly Val Ile Pro Gln Lys Val Phe Ile Phe Ser                1285 1290 1295 Gly Thr Phe Arg Lys Asn Leu Asp Pro Tyr Glu Gln Trp Ser Asp Gln            1300 1305 1310 Glu Ile Trp Lys Val Ala Asp Glu Val Gly Leu Arg Ser Val Ile Glu        1315 1320 1325 Gln Phe Pro Gly Lys Leu Asp Phe Val Leu Val Asp Gly Gly Cys Val    1330 1335 1340 Leu Ser His Gly His Lys Gln Leu Met Cys Leu Ala Arg Ser Val Leu 1345 1350 1355 1360 Ser Lys Ala Lys Ile Leu Leu Leu Asp Glu Pro Ser Ala His Leu Asp                1365 1370 1375 Pro Val Thr Tyr Gln Ile Ile Arg Arg Thr Leu Lys Gln Ala Phe Ala            1380 1385 1390 Asp Cys Thr Val Ile Leu Cys Glu His Arg Ile Glu Ala Met Leu Glu        1395 1400 1405 Cys Gln Gln Phe Leu Val Ile Glu Glu Asn Lys Val Arg Gln Tyr Asp    1410 1415 1420 Ser Ile Gln Lys Leu Leu Asn Glu Arg Ser Leu Phe Arg Gln Ala Ile 1425 1430 1435 1440 Ser Pro Ser Asp Arg Val Lys Leu Phe Pro His Arg Asn Ser Ser Lys                1445 1450 1455 Cys Lys Ser Lys Pro Gln Ile Ala Ala Leu Lys Glu Glu Thr Glu Glu            1460 1465 1470 Glu Val Gln Asp Thr Arg Leu        1475

Claims (20)

A composition comprising a guide RNA comprising two or more contiguous nucleotides complementary to a target nucleic acid sequence encoding a CFTR polypeptide,
Wherein the guide RNA comprises a polynucleotide consisting of the nucleic acid sequence of SEQ ID NO:
A composition for mutating a nucleic acid sequence encoding a CFTR polypeptide in a genome of a cell. The composition of claim 1, wherein the target nucleic acid sequence comprises the nucleic acid sequence of SEQ ID NO: 1. The polynucleotide according to claim 1, wherein the target nucleic acid sequence is a nucleotide 129 to 148, a nucleotide 119 to 138, or a nucleotide 185 to 204 from the 5 'end of SEQ ID NO: 1 in a polynucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 Composition. 2. The composition of claim 1, wherein the guide RNA is a single-chain guide RNA (sgRNA). 2. The composition of claim 1, wherein the target nucleic acid sequence comprises a protospacer adjacent motif (PAM) or a complementary sequence thereof. The PAM according to claim 5, wherein the PAM comprises a nucleic acid sequence selected from the group consisting of 5'-AGG-3 ', 5'-GGG-3', 5'-CGG-3 'and 5'-TGG-3' / RTI &gt; delete 3. The composition of claim 1, wherein the guide RNA is comprised in a vector. 9. The composition of claim 8, wherein the vector further comprises a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide. The composition according to claim 1, wherein the composition is for administration in vitro or in vivo. The composition of claim 1, further comprising a second polynucleotide comprising a nucleic acid sequence encoding a Cas polypeptide. 10. The composition of claim 9 or 11, wherein the Cas polypeptide is a Cas5 polypeptide, a Cas7 polypeptide, a Cas8 polypeptide, a Cas9 polypeptide, or a Cpf1 polypeptide. CLAIMS What is claimed is: 1. A method of mutating a nucleic acid sequence encoding a CFTR polypeptide in a cell's genome, comprising incubating the cell with the composition of claim 1. 14. The method of claim 13, wherein the CFTR polypeptide expression is suppressed or increased relative to the negative control by mutating the nucleic acid sequence. Wherein the nucleic acid sequence encoding the CFTR polypeptide has been mutated and has been deposited with Accession No. KCLRF-BP-00388. delete delete delete Incubating the cell of claim 15 with a test compound;
Measuring CFTR polypeptide activity in the incubated cells; And
Selecting a test compound having increased or decreased measured CFTR polypeptide activity relative to a negative control,
A method of screening a test compound that increases or decreases CFTR polypeptide activity. 21. The method of claim 19, wherein the screened test compound is selected from the group consisting of cystic fibrosis, chronic obstructive pulmonary disease (COPD), dry eye disease, bronchitis, emphysema, bronchiectasis, asthma, sinusitis, Sjogren's syndrome, Wherein the compound is a candidate compound for the prophylaxis or treatment of a disease selected from the group consisting of Marfan syndrome, Fabry disease, Kocher disease, pigmented retinitis, Alzheimer's disease, type II diabetes, Parkinson's disease and Creutzfeldt-Jakob disease.

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