CELL LINE WHICH IS KNOCK OUT THE BMP RECEPTOR GENES AND A METHOD OF PRODUCING TARGET PROTEINS USING THE SAME

Abstract

The present invention relates to a transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII is knocked out. The BMP type I receptor BMPRIA or BMP type II receptor BMPRII gene which plays an important role in intracellular signal transduction in CHO cells is knocked out to prevent the activation of self concentration control pathway and the signal transduction mediated by BMP in CHO cells, so that the productivity of a target protein to be produced can be improved.

Claims

1. A transgenic CHO cell line wherein a bone morphogenetic protein (BMP) type I receptor gene (BMPRIA) or a BMP type II receptor gene (BMPRII) is knocked out.

2. The CHO cell line according to claim 1, wherein the cell line has a mutation in the 2.sup.nd exon nucleotide sequence of the BMPRIA gene.

3. The CHO cell line according to claim 1, wherein the cell line has a mutation in the 2.sup.nd to 5.sup.th exon nucleotide sequences of the BMPRII gene.

4. A method for preparing a transgenic CHO cell line, wherein the BMP receptor gene BMPRIA or BMPRII is knocked-out, the method comprising the following steps: 1) constructing a vector to knock-out the BMP receptor gene BMPRIA or BMPRII; 2) introducing the vector of step 1) into a CHO cell line; and 3) selecting the cell line in which the BMP receptor gene BMPRIA or BMPRII has been knocked-out from those CHO cell lines introduced with the vector of step 2).

5. The method according to claim 4, wherein the vector of step 1) includes gene scissors.

6. The method according to claim 4, wherein the vector that knocks out the BMPRIA gene of step 1) is a vector comprising the nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRIA gene or a vector comprising the nucleotide sequence encoding Cas9 protein.

7. The method according to claim 4, wherein the vector that knocks out the BMPRII gene of step 1) is a vector comprising the nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRII gene, a vector comprising the nucleotide sequence of sgRNA targeting the 5.sup.th exon nucleotide sequence of BMPRII gene or a vector comprising the nucleotide sequence encoding Cas9 protein.

8. A method of producing a target protein comprising the following steps: 1) introducing a vector containing the nucleotide sequence encoding a target protein into the transgenic CHO cell line of claim 1, wherein the BMP receptor gene BMPRIA or BMPRII has been knocked-out; 2) culturing the cell line prepared in step 1) above; and 3) separating and purifying the target protein produced in step 2) above.

9. The method according to claim 8, wherein the CHO cell line of step 1) is a dihydrofolate reductase (DHFR) gene knock out cell line.

10. The method according to claim 8, wherein the vector of step 1) additionally includes a nucleotide sequence encoding DHFR protein.

11. The method according to claim 8, wherein the culture in step 2) is performed by treating methotrexate (MTX).

12. The method according to claim 8, wherein the target protein is selected from the group consisting of BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14 and BMP15.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0025] FIG. 1 is a diagram illustrating the structures of BMPRIA gene and BMPRII gene in CHO cells, the location of sgRNA targeting each BMP receptor gene and the primer binding sites of junction PCR and out-out PCR.

[0026] FIG. 2 is a diagram illustrating the nucleotide sequences of knock-out target sequences in the BMPRIA gene knock-out clones (22, 35, 43, 47, 61 and 68).

[0027] FIG. 3 is a set of photographs illustrating the results of junction PCR and out-out PCR to confirm the BMPRII gene knock-out clones (20, 52 and 118)

[0028] (wt: wild type;

[0029] e5-6: PCR with primers targeting exons 5 to 6;

[0030] e2-3: PCR with primers targeting exons 2 to 3;

[0031] e2-5: PCR with primers targeting exons 2 to 5).

[0032] FIG. 4 is a graph illustrating the concentration of rhBMP4 produced in the BMPRIA or BMPRII gene knock-out cell line

[0033] (DG44 wt: wild type (CHO DG44 host cells);

[0034] IA-KO-47: clone 47 with BMPRIA gene knock-out;

[0035] II-KO-20: clone 20 with BMPRII gene knock-out;

[0036] II-KO-118: clone 118 with BMPRII gene knock-out).

[0037] FIG. 5 is a graph illustrating the maximum production of rhBMP4 in the BMPRIA or BMPRII gene knock-out cell line

[0038] (DG44 wt: wild type (CHO DG44 host cells);

[0039] KO: IA-KO-47, II-KO-20 and II-KO-118).

[0040] FIG. 6 is a graph illustrating the cell concentration of the BMPRIA or BMPRII gene knock-out cell line expressing recombinant human BMP4 (rhBMP4)

[0041] (DG44 wt: wild type (CHO DG44 host cells);

[0042] KO: IA-KO-47, II-KO-20 and II-KO-118).

[0043] FIG. 7 is a graph illustrating the maximum production of rhBMP4 in suspension culture of the BMPRIA or BMPRII gene knock-out cell line expressing recombinant human BMP4 (rhBMP4)

[0044] (DG44 wt: wild type (CHO DG44 host cells);

[0045] KO: IA-KO-47, II-KO-20 and II-KO-118).

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] Hereinafter, the present invention is described in detail.

[0047] The present invention provides a transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII is knocked-out.

[0048] The said BMPRIA gene is a BMP type I receptor gene and the said BMPRII gene is a BMP type II receptor gene.

[0049] Particularly, the cell line above can have a mutation in the nucleotide sequence of the 2.sup.nd exon of the BMPRIA gene. More particularly, the cell line can have a mutation in the nucleotide sequence 5-ATGCATGTGTTATTAATAGCATCATCTGGGCAGTGGCCTGAGCAGTAACACTTTAAGAA AGGCAAAGTATCCTCCGGGGCTAAAGTCACTCCATTTTCTGGCTTCTTCTGGTCCAAGT CTGATTTCATACCAGTACCATGGAGCATACTGTCTAGATTCTGCC-3 (SEQ. ID. NO: 1) of the 2.sup.nd exon of the BMPRIA gene. More precisely, the BMPRIA gene knock out transgenic CHO cell line has been transfected with a vector comprising the nucleotide sequences represented by SEQ. ID. NO: 10 and SEQ. ID. NO: 13, so that the nucleotide sequence of the 2.sup.nd exon of the BMPRIA gene has been mutated.

[0050] In addition, the cell line can have a mutation in the nucleotide sequences of the 2.sup.nd to 5.sup.th exons of the BMPRII gene. More particularly the cell line can have a mutation in the nucleotide sequences 5-CTTCCCAGAATCAAGAACGGCTGTGTGCATTTAAAGATCCCTACCAGCAAGACCTTGGG ATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCCAAAGGTAGCAC ATGCTATGGTCTATGGGAGAAATCAAAAGGGGACATCAATCTTGTGAAACAAG-3 (SEQ. ID. NO: 2) of the 2.sup.nd exon, 5-GATGTTGGTCTCACATTGGCGATCCTCAAGAGTGTCACTATGAAGAATGTGTAGTAACT ACTACCCCACCCTCAATTCAGAATGGAACATACCGTTTTTGCTGCTGTAGTACAGATTT ATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGATACAACACCACTCA-3 (SEQ. ID. NO: 3) of the 3rd exon, 5-GTCCACCTCATTCATTTAATCGAGATGAGACAATAATCATTGCTTTGGCATCAGTCTCT GTATTAGCTGTTTTGATAGTCGCCTTATGTTTTGGATACAGAATGTTGACAG-3 (SEQ. ID. NO: 4) of the exon and 5-GAGACCGAAAACAAGGCCTTCACAGTATGAACATGATGGAAGCAGCGGCGTCAGAGCCT TCTCTGGACTTGGATAATCTGAAGCTGCTGGAG-3 (SEQ. ID. NO: 5) of the 5.sup.th exon of the BMPRII gene. More precisely, the BMPRII gene knock out transgenic CHO cell line has been transfected with a vector comprising the nucleotide sequences represented by SEQ. ID. NO: 11, SEQ. ID. NO: 12 and SEQ. ID. NO: 13, so that the nucleotide sequences of the 2.sup.nd to 5.sup.th exons of the BMPRII gene have been mutated.

[0051] The CHO cell line above can be a DHFR (dihydrofolate reductase) gene knock out cell line. The expression of a target protein gene in the DHFR gene knock out CHO cell line can be amplified by using DHFR/MTX (dihydrofolate reductase/methotrexate) system. Particularly, a vector comprising the nucleotide sequence encoding DHFR and a target protein is introduced into the host cells in which DFHR gene necessary for cell growth is knocked-out. Then, the cells are treated with MTX suppressing DHFR gene expression to inhibit cell growth. At this time, the cells can amplify the expression of a gene encoding DHFR and a target protein in order to overcome the suppression above.

[0052] The present invention also provides a preparation method of a transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII is knocked-out. The preparation method comprises the following steps:

[0053] 1) constructing a vector to knock-out the BMP receptor gene BMPRIA or BMPRII;

[0054] 2) introducing the vector of step 1) into a CHO cell line; and

[0055] 3) selecting the CHO cell line in which the BMP receptor gene BMPRIA or BMPRII has been knocked-out from those CHO cell lines introduced with the vector of step 2).

[0056] The said BMPRIA gene is a BMP type I receptor gene and the said BMPRII gene is a BMP type II receptor gene. Particularly, the BMPRIA gene of step 1) can have a mutation in the nucleotide sequence of the 2.sup.nd exon of the BMPRIA gene. More particularly, the BMPRIA gene can have a mutation in the nucleotide sequence 5-ATGCATGTGTTATTAATAGCATCATCTGGGCAGTGGCCTGAGCAGTAACACTTTAAGAA AGGCAAAGTATCCTCCGGGGCTAAAGTCACTCCATTTTCTGGCTTCTTCTGGTCCAAGT CTGATTTCATACCAGTACCATGGAGCATACTGTCTAGATTCTGCC-3 (SEQ. ID. NO: 1) of the 2.sup.nd exon of the BMPRIA gene. In addition, the BMPRII gene of step 1) can have a mutation in the nucleotide sequences of the 2.sup.nd to 5.sup.th exons of the BMPRII gene. More particularly, the BMPRII gene can have a mutation in the nucleotide sequences 5-CTTCCCAGAATCAAGAACGGCTGTGTGCATTTAAAGATCCCTACCAGCAAGACCTTGGG ATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCCAAAGGTAGCAC ATGCTATGGTCTATGGGAGAAATCAAAAGGGGACATCAATCTTGTGAAACAAG-3 (SEQ. ID. NO: 2) of the 2.sup.nd exon, 5-GATGTTGGTCTCACATTGGCGATCCTCAAGAGTGTCACTATGAAGAATGTGTAGTAACT ACTACCCCACCCTCAATTCAGAATGGAACATACCGTTTTTGCTGCTGTAGTACAGATTT ATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGATACAACACCACTCA-3 (SEQ. ID. NO: 3) of the 3.sup.rd exon, 5-GTCCACCTCATTCATTTAATCGAGATGAGACAATAATCATTGCTTTGGCATCAGTCTCT GTATTAGCTGTTTTGATAGTCGCCTTATGTTTTGGATACAGAATGTTGACAG-3

[0057] (SEQ. ID. NO: 4) of the 4.sup.th exon and 5-GAGACCGAAAACAAGGCCTTCACAGTATGAACATGATGGAAGCAGCGGCGTCAGAGCCT TCTCTGGACTTGGATAATCTGAAGCTGCTGGAG-3 (SEQ. ID. NO: 5) of the 5.sup.th exon of the BMPRII gene.

[0058] The vector of step 1) preferably includes gene scissors, and the gene scissors are preferably selected from the group consisting of ZFN (zinc-finger nuclease), TALEN (transcription activator-like effector nuclease) and CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein-9), but not always limited thereto. According to an example of the present invention, the vector can include CRISPR/Case9, and particularly the vector can be the one that can express sgRNA and Cas9 protein targeting the BMP receptor gene BMPRIA or BMPRII.

[0059] More particularly, the vector that can knock out the BMPRIA gene of step 1) can be a vector containing the nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRIA gene or a vector containing the nucleotide sequence encoding Cas9 protein. According to an example of the present invention, the Cas9 protein is composed of the amino acid sequence represented by SEQ. ID. NO: 9. The nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRIA gene can be 5-GAAAGGCAAAGTATCCTCCGGGG-3 (SEQ. ID. NO: 6). More particularly, the vector that can knock out the BMPRIA gene of step 1) can be composed of the nucleotide sequence represented by SEQ. ID. NO: 10 or SEQ. ID. NO: 13.

[0060] The vector that can knock out the BMPRII gene of step 1) can be a vector comprising the nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRII gene, a vector comprising the nucleotide sequence of sgRNA targeting the 5.sup.th exon nucleotide sequence of BMPRII gene or a vector comprising the nucleotide sequence encoding Cas9 protein. The nucleotide sequence of sgRNA targeting the 2.sup.nd exon nucleotide sequence of BMPRII gene can be 5-GGGACAATATTATGCTCCAAAGG-3 (SEQ. ID. NO: 7). The nucleotide sequence of sgRNA targeting the 5.sup.th exon nucleotide sequence of BMPRII gene can be 5-AGCGGCGTCAGAGCCTTCTCTGG-3 (SEQ. ID. NO: 8). More particularly, the vector that can knock out the BMPRII gene of step 1) can be composed of the nucleotide sequence represented by SEQ. ID. NO: 11, SEQ. ID. NO: 12 or SEQ. ID. NO: 13.

[0061] A method for introducing the vector of step 2) into the CHO cell line is exemplified by transformation, transfection, electroporation, transduction, microinjection or ballistic introduction, but not always limited thereto. According to an example of the present invention, the vector of step 2) can be introduced into the CHO cell line via transformation using lipofectamine.

[0062] The CHO cell line of step 2) can be a DHFR gene knock out cell line. The expression of a target protein gene in the DHFR gene knock out CHO cell line can be amplified by using DHFR/MTX system.

[0063] In addition, the step of selecting the cell line in which the BMP receptor gene BMPRIA or BMPRII has been knocked-out in step 3) can be achieved by various methods informed to those in the art. For example, PCR using genomic DNA as a template or nucleotide sequencing can be used.

[0064] In a preferred embodiment of the present invention, the present inventors introduced a vector expressing Cas 9 protein and sgRNA targeting BMPRIA gene or BMPRII gene in CHO host cells and then selected the cell line in which the BMPRIA gene or BMPRII gene was mutated or deleted in all homologous chromosome pairs in CHO cells (see FIGS. 1-3) .

[0065] In addition, the present invention provides a production method of a target protein using a transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII is knocked-out. The production method of a target protein comprises the following steps:

[0066] 1) introducing a vector containing the nucleotide sequence encoding a target protein into the transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII has been knocked-out;

[0067] 2) culturing the cell line prepared in step 1) above; and

[0068] 3) separating and purifying the target protein produced in step 2) above.

[0069] The transgenic CHO cell line in which the BMP receptor gene BMPRIA or BMPRII has been knocked-out of step 1) is as described above. According to an example of the present invention, the BMPRIA gene of step 1) can have a mutation in the nucleotide sequence of the 2.sup.nd exon of the BMPRIA gene. More particularly, the BMPRIA gene can have a mutation in the nucleotide sequence 5-ATGCATGTGTTATTAATAGCATCATCTGGGCAGTGGCCTGAGCAGTAACACTTTAAGAA AGGCAAAGTATCCTCCGGGGCTAAAGTCACTCCATTTTCTGGCTTCTTCTGGTCCAAGT CTGATTTCATACCAGTACCATGGAGCATACTGTCTAGATTCTGCC-3 (SEQ. ID. NO: 1) of the 2.sup.nd exon of the BMPRIA gene. In addition, the BMPRII gene of step 1) can have a mutation in the nucleotide sequences of the 2.sup.nd to 5.sup.th exons of the BMPRII gene. More particularly, the BMPRII gene can have a mutation in the nucleotide sequences 5-CTTCCCAGAATCAAGAACGGCTGTGTGCATTTAAAGATCCCTACCAGCAAGACCTTGGG ATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAATATTATGCTCCAAAGGTAGCAC ATGCTATGGTCTATGGGAGAAATCAAAAGGGGACATCAATCTTGTGAAACAAG-3 (SEQ. ID. NO: 2) of the 2nd exon, 5-GATGTTGGTCTCACATTGGCGATCCTCAAGAGTGTCACTATGAAGAATGTGTAGTAACT ACTACCCCACCCTCAATTCAGAATGGAACATACCGTTTTTGCTGCTGTAGTACAGATTT ATGTAATGTCAACTTTACTGAGAATTTTCCACCTCCTGATACAACACCACTCA-3 (SEQ. ID. NO: 3) of the 3.sup.rd exon, 5-GTCCACCTCATTCATTTAATCGAGATGAGACAATAATCATTGCTTTGGCATCAGTCTCT GTATTAGCTGTTTTGATAGTCGCCTTATGTTTTGGATACAGAATGTTGACAG-3 (SEQ. ID. NO: 4) of the 4.sup.th exon and 5-GAGACCGAAAACAAGGCCTTCACAGTATGAACATGATGGAAGCAGCGGCGTCAGAGCCT TCTCTGGACTTGGATAATCTGAAGCTGCTGGAG-3 (SEQ. ID. NO: 5) of the 5.sup.th exon of the BMPRII gene.

[0070] The CHO cell line of step 1) can be a DHFR gene knock out cell line. The expression of a target protein gene in the DHFR gene knock out CHO cell line can be amplified by using DHFR/MTX system. Particularly, a vector comprising the nucleotide sequence encoding DHFR and a target protein is introduced into the host cells in which DFHR gene necessary for cell growth is knocked-out. Then, the cells are treated with MTX suppressing DHFR gene expression to inhibit cell growth. At this time, the cells can amplify the expression of a gene encoding DHFR and a target protein in order to overcome the suppression above. So, the vector of step 1) can additionally include a nucleotide sequence encoding DHFR protein.

[0071] The target protein of step 1) can be any one selected from the group consisting of BMP2, BMP3, BMP4, BMPS, BMP6, BMP7, BMP8, BMP9, BMP10, BMP11, BMP12, BMP13, BMP14 and BMP15, but not always limited thereto.

[0072] A method for introducing the vector comprising the nucleotide sequence encoding the target protein of step 1) into the CHO cell line is exemplified by transformation, transfection, electroporation, transduction, microinjection or ballistic introduction, but not always limited thereto. According to an example of the present invention, the vector of step 1) can be introduced into the CHO cell line via transformation using lipofectamine.

[0073] The cell line of step 2) can be cultured by batch culture, fed-batch culture or continuous culture, but not always limited thereto. The culture in step 2) is preferably performed after treating MTX (methotrexate).

[0074] In a preferred embodiment of the present invention, the present inventors constructed a CHO cell line in which BMPRIA or BMPRII gene was knocked out, and introduced a vector comprising the nucleotide sequence encoding DHFR and BMP4 (recombinant human BMP4, rhBMP4) into the cell line. The cell line was treated with MTX, followed by culture. Then, rhBMP4 production was confirmed. As a result, it was confirmed that the growth of CHO cells in which BMPRIA or BMPRII gene was knocked out was increased and the production of rhBMP4 was higher than the wild type (see FIGS. 4-7).

[0075] Therefore, the BMP receptor gene knock out CHO cell line of the present invention can prevent the activation of intracellular signal transduction and self concentration control pathway to increase CHO cell growth, so that the productivity of a target protein to be produced can be improved using the cell line.

[0076] Practical and presently preferred embodiments of the present invention are illustrative as shown in the following Examples.

[0077] However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modifications and improvements within the spirit and scope of the present invention.

Example 1: Construction of BMPRIA Gene Knock Out CHO Cell Line

[0078] To construct a BMPRIA gene knock out CHO cell line, CHO DG44 host cells were cultured in IMDM (Iscov's Modified Dulbecco's Medium) supplemented with 7% (v/v) dFBS (dialyzed fetal bovine serum) and hypoxanthine/thymidine (HT) via adhesion culture. A vector containing gene scissors targeting the 2.sup.nd exon (SEQ. ID. NO: 1) of BMPRIA gene was introduced into the cultured CHO cells. Particularly, the said vector was the one expressing Cas9 protein (SEQ. ID. NO: 9) and sgRNA (SEQ. ID. NO: 6) targeting the 2.sup.nd exon sequence of BMPRIA gene. The vector was prepared by Toolgene. The entire sequence of the constructed vector was identified as SEQ. ID. NO: 10 and SEQ. ID. NO: 13. The constructed vector was introduced into CHO cells using lipofectamine (lipofectamine 2000, Life technology).

Example 2: Construction of BMPRII Gene Knock Out CHO Cell Line

[0079] To construct a BMPRII gene knock out CHO cell line, cells were cultured by the same manner as described in Example 1. A vector containing gene scissors targeting the 2.sup.nd to 5.sup.th exons (SEQ. ID. NOs: 2-5) of BMPRII gene was introduced into the cultured CHO cells. Particularly, the said vector includes the nucleotide sequences expressing sgRNA (SEQ. ID. NO: 7) targeting the 2.sup.nd exon sequence of BMPRII gene, sgRNA (SEQ. ID. NO: 8) targeting the 5.sup.th exon sequence of BMPRII gene and Cas9 protein (SEQ. ID. NO: 9). Using the sgRNA targeting the 2.sup.nd and 5.sup.th exons of BMPRII gene, all of the genomic fragments between the 2.sup.nd and 5.sup.th exons of BMPRII gene were removed. The vector was prepared by Toolgene. The entire sequence of the constructed vector was identified as SEQ. ID. NO: 11, SEQ. ID. NO: 12 and SEQ. ID. NO: 13. The constructed vector was introduced into CHO cells using lipofectamine (lipofectamine 2000, Life technology).

TABLE-US-00001 TABLEA SEQ. ID. Geneor NucleotideSequence(5.fwdarw.3)or NO: protein Aminoacidsequence 1 2.sup.ndexonsof ATGCATGTGTTATTAATAGCATCATCTGGGCAGTGGCCTGAGCAGTAA BMPRIAgene CACTTTAAGAAAGGCAAAGTATCCTCCGGGGCTAAAGTCACTCCATTT TCTGGCTTCTTCTGGTCCAAGTCTGATTTCATACCAGTACCATGGAGC ATACTGTCTAGATTCTGCC 2 2.sup.ndexonsof CTTCCCAGAATCAAGAACGGCTGTGTGCATTTAAAGATCCCTACCAGC BMPRIIgene AAGACCTTGGGATAGGTGAGAGTAGAATCTCTCATGAAAATGGGACAA TATTATGCTCCAAAGGTAGCACATGCTATGGTCTATGGGAGAAATCAA AAGGGGACATCAATCTTGTGAAACAAG 3 3.sup.rdexonsof GATGTTGGTCTCACATTGGCGATCCTCAAGAGTGTCACTATGAAGAAT BMPRIIgene GTGTAGTAACTACTACCCCACCCTCAATTCAGAATGGAACATACCGTT TTTGCTGCTGTAGTACAGATTTATGTAATGTCAACTTTACTGAGAATT TTCCACCTCCTGATACAACACCACTCA 4 4.sup.thexonsof GTCCACCTCATTCATTTAATCGAGATGAGACAATAATCATTGCTTTGG BMPRIIgene CATCAGTCTCTGTATTAGCTGTTTTGATAGTCGCCTTATGTTTTGGAT ACAGAATGTTGACAG 5 5.sup.thexonsof GAGACCGAAAACAAGGCCTTCACAGTATGAACATGATGGAAGCAGCGG BMPRIIgene CGTCAGAGCCTTCTCTGGACTTGGATAATCTGAAGCTGCTGGAG 6 sgRNA GAAAGGCAAAGTATCCTCCGGGG targeting the2.sup.ndexon sequenceof BMPRIAgene 7 sgRNA GGGACAATATTATGCTCCAAAGG targeting the2.sup.ndexon sequenceof BMPRIIgene 8 sgRNA AGCGGCGTCAGAGCCTTCTCTGG targeting the5.sup.thexon sequenceof BMPRIAgene 9 Casprotein MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLI GALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDS FEHRLEESELVEEDKKHERHPIEGNIVDEVAYHEKYPTIYHLRKKLVD STDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTY NQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGN LIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYAD LFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLK ALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMD GTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPF LKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEE VVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLT LFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRD KQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSL HEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQ TTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYL QNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNR GKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELD KAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKS KLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEF VYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGE IRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGG FSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKG KSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPK YSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGS PEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNK HRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLD ATLIHQSITGLYETRIDLSQLGGD 10 Avector gacgaagactcaattgtcgattagtgaacggatctcgacggtatcgat expressing cacgagactagcctcgagcggccgcccccttcaccgagggcctatttc sgRNAthat ccatgattccttcatatttgcatatacgatacaaggctgttagagaga targetsthe taattggaattaatttgactgtaaacacaaagatattagtacaaaata 2.sup.ndexon cgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaat sequenceof tatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatt BMPRIAgene tcgatttcttggctttatatatcttgtggaaaggacgaaacaccgGAA AGGCAAAGTATCCTCCGGGGgttttagagctagaaatagcaagttaaa ataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc ttttttctagattcgcgatgtacgggccagatatacgcgttgacattg attattgactagttgtcttcctgcattaatgaatcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact caaaggcggtaatacggttatccacagaatcaggggataacgcaggaa agaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg gaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcgg tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc cggtaagacacgacttatcgccactggcagcagccactggtaacagga ttagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt ggcctaactacggctacactagaaggacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccg gcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt tggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct gtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgatac cgcgagatccacgctcaccggctccagatttatcagcaataaaccagc cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct ccatccagtctattaattgttgccgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtgg tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgt tatcactcatggttatggcagcactgcataattctcttactgtcatgc catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa tacgggataataccgcgccacatagcagaactttaaaagtgctcatca ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgt tgagatccagttcgatgtaacccactcgtgcacccaactgatcttcag catcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggc aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac tcatactcttcctttttcaatattattgaagcatttatcagggttatt gtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaa taggggttccgcgcacatttccccgaaaagtgccacctgacgtc 11 Avector gacgaagactcaattgtcgattagtgaacggatctcgacggtatcgat expressing cacgagactagcctcgagcggccgcccccttcaccgagggcctatttc sgRNAthat ccatgattccttcatatttgcatatacgatacaaggctgttagagaga targetsthe taattggaattaatttgactgtaaacacaaagatattagtacaaaata 2.sup.ndexon cgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaat sequenceof tatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatt BMPRIIgene tcgatttcttggctttatatatcttgtggaaaggacgaaacaccgGGG ACAATATTATGCTCCAAAGGgttttagagctagaaatagcaagttaaa ataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc ttttttctagattcgcgatgtacgggccagatatacgcgttgacattg attattgactagttgtcttcctgcattaatgaatcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact caaaggcggtaatacggttatccacagaatcaggggataacgcaggaa agaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg gaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcgg tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc cggtaagacacgacttatcgccactggcagcagccactggtaacagga ttagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt ggcctaactacggctacactagaaggacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccg gcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt tggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct gtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgatac cgcgagatccacgctcaccggctccagatttatcagcaataaaccagc cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct ccatccagtctattaattgttgccgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtgg tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgt tatcactcatggttatggcagcactgcataattctcttactgtcatgc catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa tacgggataataccgcgccacatagcagaactttaaaagtgctcatca ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgt tgagatccagttcgatgtaacccactcgtgcacccaactgatcttcag catcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggc aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac tcatactcttcctttttcaatattattgaagcatttatcagggttatt gtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaa taggggttccgcgcacatttccccgaaaagtgccacctgacgtc 12 Avector gacgaagactcaattgtcgattagtgaacggatctcgacggtatcgat expressing cacgagactagcctcgagcggccgcccccttcaccgagggcctatttc sgRNAthat ccatgattccttcatatttgcatatacgatacaaggctgttagagaga targetsthe taattggaattaatttgactgtaaacacaaagatattagtacaaaata 5.sup.thexon cgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaat sequenceof tatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatt BMPRIIgene tcgatttcttggctttatatatcttgtggaaaggacgaaacaccgAGC GGCGTCAGAGCCTTCTCTGGgttttagagctagaaatagcaagttaaa ataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgc ttttttctagattcgcgatgtacgggccagatatacgcgttgacattg attattgactagttgtcttcctgcattaatgaatcggccaacgcgcgg ggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactg actcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcact caaaggcggtaatacggttatccacagaatcaggggataacgcaggaa agaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaagg ccgcgttgctggcgtttttccataggctccgcccccctgacgagcatc acaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactat aaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg gaagcgtggcgctttctcaatgctcacgctgtaggtatctcagttcgg tgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttc agcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacc cggtaagacacgacttatcgccactggcagcagccactggtaacagga ttagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggt ggcctaactacggctacactagaaggacagtatttggtatctgcgctc tgctgaagccagttaccttcggaaaaagagttggtagctcttgatccg gcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagc agattacgcgcagaaaaaaaggatctcaagaagatcctttgatctttt ctacggggtctgacgctcagtggaacgaaaactcacgttaagggattt tggtcatgagattatcaaaaaggatcttcacctagatccttttaaatt aaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggt ctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatct gtctatttcgttcatccatagttgcctgactccccgtcgtgtagataa ctacgatacgggagggcttaccatctggccccagtgctgcaatgatac cgcgagatccacgctcaccggctccagatttatcagcaataaaccagc cagccggaagggccgagcgcagaagtggtcctgcaactttatccgcct ccatccagtctattaattgttgccgggaagctagagtaagtagttcgc cagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtgg tgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaac gatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggtta gctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgt tatcactcatggttatggcagcactgcataattctcttactgtcatgc catccgtaagatgcttttctgtgactggtgagtactcaaccaagtcat tctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaa tacgggataataccgcgccacatagcagaactttaaaagtgctcatca ttggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgt tgagatccagttcgatgtaacccactcgtgcacccaactgatcttcag catcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggc aaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatac tcatactcttcctttttcaatattattgaagcatttatcagggttatt gtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaa taggggttccgcgcacatttccccgaaaagtgccacctgacgtc 13 Avector GACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAA expressing TCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTG Casprotein TGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAA GGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGC GTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATT GATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTC ATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGA CGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAAT GGGTGGACTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGT ATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGC CCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGT TTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGAT TTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC AAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGA CGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAG CTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTA ATACGACTCACTATAGGGAGACCCAAGCTTGCCACCATGGACAAGAAG TACAGCATCGGCCTGGACATCGGTACCAACAGCGTGGGCTGGGCCGTG ATCACCGACGAGTACAAGGTGCCCAGCAAGAAGTTCAAGGTGCTGGGC AACACCGACCGCCACAGCATCAAGAAGAACCTGATCGGCGCCCTGCTG TTCGACAGCGGCGAGACCGCCGAGGCCACCCGCCTGAAGCGCACCGCC CGCCGCCGCTACACCCGCCGCAAGAACCGCATCTGCTACCTGCAGGAG ATCTTCAGCAACGAGATGGCCAAGGTGGACGACAGCTTCTTCCACCGC CTGGAGGAGAGCTTCCTGGTGGAGGAGGACAAGAAGCACGAGCGCCAC CCCATCTTCGGCAACATCGTGGACGAGGTGGCCTACCACGAGAAGTAC CCCACCATCTACCACCTGCGCAAGAAGCTGGTGGACAGCACCGACAAG GCCGACCTGCGCCTGATCTACCTGGCCCTGGCCCACATGATCAAGTTC CGCGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGAC GTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTC GAGGAGAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTG AGCGCCCGCCTGAGCAAGAGCCGCCGCCTGGAGAACCTGATCGCCCAG CTGCCCGGCGAGAAGAAGAACGGCCTGTTCGGCAACCTGATCGCCCTG AGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCCGAG GACGCCAAGCTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGAC AACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTCCTGGCC GCCAAGAACCTGAGCGACGCCATCCTGCTGAGCGACATCCTGCGCGTG AACACCGAGATCACCAAGGCCCCCCTGAGCGCCAGCATGATCAAGCGC TACGACGAGCACCACCAGGACCTGACCCTGCTGAAGGCCCTGGTGCGC CAGCAGCTGCCCGAGAAGTACAAGGAGATCTTCTTCGACCAGAGCAAG AACGGCTACGCCGGCTACATCGACGGCGGCGCCAGCCAGGAGGAGTTC TACAAGTTCATCAAGCCCATCCTGGAGAAGATGGACGGCACCGAGGAG CTGCTGGTGAAGCTGAACCGCGAGGACCTGCTGCGCAAGCAGCGCACC TTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGCGAGCTGCAC GCCATCCTGCGCCGCCAGGAGGACTTCTACCCCTTCCTGAAGGACAAC CGCGAGAAGATCGAGAAGATCCTGACCTTCCGCATCCCCTACTACGTG GGCCCCCTGGCCCGCGGCAACAGCCGCTTCGCCTGGATGACCCGCAAG AGCGAGGAGACCATCACCCCCTGGAACTTCGAGGAGGTGGTGGACAAG GGCGCCAGCGCCCAGAGCTTCATCGAGCGCATGACCAACTTCGACAAG AACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAG TACTTCACCGTGTACAACGAGCTGACCAAGGTGAAGTACGTGACCGAG GGCATGCGCAAGCCCGCCTTCCTGAGCGGCGAGCAGAAGAAGGCCATC GTGGACCTGCTGTTCAAGACCAACCGCAAGGTGACCGTGAAGCAGCTG AAGGAGGACTACTTCAAGAAGATCGAGTGCTTCGACAGCGTGGAGATC AGCGGCGTGGAGGACCGCTTCAACGCCAGCCTGGGCACCTACCACGAC CTGCTGAAGATCATCAAGGACAAGGACTTCCTGGACAACGAGGAGAAC GAGGACATCCTGGAGGACATCGTGCTGACCCTGACCCTGTTCGAGGAC CGCGAGATGATCGAGGAGCGCCTGAAGACCTACGCCCACCTGTTCGAC GACAAGGTGATGAAGCAGCTGAAGCGCCGCCGCTACACCGGCTGGGGC CGCCTGAGCCGCAAGCTTATCAACGGCATCCGCGACAAGCAGAGCGGC AAGACCATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCAACCGCAAC TTCATGCAGCTGATCCACGACGACAGCCTGACCTTCAAGGAGGACATC CAGAAGGCCCAGGTGAGCGGCCAGGGCGACAGCCTGCACGAGCACATC GCCAACCTGGCCGGCAGCCCCGCCATCAAGAAGGGCATCCTGCAGACC GTGAAGGTGGTGGACGAGCTGGTGAAGGTGATGGGCCGCCACAAGCCC GAGAACATCGTGATCGAGATGGCCCGCGAGAACCAGACCACCCAGAAG GGCCAGAAGAACAGCCGCGAGCGCATGAAGCGCATCGAGGAGGGCATC AAGGAGCTGGGCAGCCAGATCCTGAAGGAGCACCCCGTGGAGAACACC CAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAACGGCCGC GACATGTACGTGGACCAGGAGCTGGACATCAACCGCCTGAGCGACTAC GACGTGGACCACATCGTGCCCCAGAGCTTCCTGAAGGACGACAGCATC GACAACAAGGTGCTGACCCGCAGCGACAAGAACCGCGGCAAGAGCGAC AACGTGCCCAGCGAGGAGGTGGTGAAGAAGATGAAGAACTACTGGCGC CAGCTGCTGAACGCCAAGCTGATCACCCAGCGCAAGTTCGACAACCTG ACCAAGGCCGAGCGCGGCGGCCTGAGCGAGCTGGACAAGGCCGGCTTC ATCAAGCGCCAGCTGGTGGAGACCCGCCAGATCACCAAGCACGTGGCC CAGATCCTGGACAGCCGCATGAACACCAAGTACGACGAGAACGACAAG CTGATCCGCGAGGTGAAGGTGATCACCCTGAAGAGCAAGCTGGTGAGC GACTTCCGCAAGGACTTCCAGTTCTACAAGGTGCGCGAGATCAACAAC TACCACCACGCCCACGACGCCTACCTGAACGCCGTGGTGGGCACCGCC CTGATCAAGAAGTACCCCAAGCTGGAGAGCGAGTTCGTGTACGGCGAC TACAAGGTGTACGACGTGCGCAAGATGATCGCCAAGAGCGAGCAGGAG ATCGGCAAGGCCACCGCCAAGTACTTCTTCTACAGCAACATCATGAAC TTCTTCAAGACCGAGATCACCCTGGCCAACGGCGAGATCCGCAAGCGC CCCCTGATCGAGACCAACGGCGAGACCGGCGAGATCGTGTGGGACAAG GGCCGCGACTTCGCCACCGTGCGCAAGGTGCTGAGCATGCCCCAGGTG AACATCGTGAAGAAGACCGAGGTGCAGACCGGCGGCTTCAGCAAGGAG AGCATCCTGCCCAAGCGCAACAGCGACAAGCTGATCGCCCGCAAGAAG GACTGGGACCCCAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCC TACAGCGTGCTGGTGGTGGCCAAGGTGGAGAAGGGCAAGAGCAAGAAG CTGAAGAGCGTGAAGGAGCTGCTGGGCATCACCATCATGGAGCGCAGC AGCTTCGAGAAGAACCCCATCGACTTCCTGGAGGCCAAGGGCTACAAG GAGGTGAAGAAGGACCTGATCATCAAGCTGCCCAAGTACAGCCTGTTC GAGCTGGAGAACGGCCGCAAGCGCATGCTGGCCAGCGCCGGCGAGCTG CAGAAGGGCAACGAGCTGGCCCTGCCCAGCAAGTACGTGAACTTCCTG TACCTGGCCAGCCACTACGAGAAGCTGAAGGGCAGCCCCGAGGACAAC GAGCAGAAGCAGCTGTTCGTGGAGCAGCACAAGCACTACCTGGACGAG ATCATCGAGCAGATCAGCGAGTTCAGCAAGCGCGTGATCCTGGCCGAC GCCAACCTGGACAAGGTGCTGAGCGCCTACAACAAGCACCGCGACAAG CCCATCCGCGAGCAGGCCGAGAACATCATCCACCTGTTCACCCTGACC AACCTGGGCGCCCCCGCCGCCTTCAAGTACTTCGACACCACCATCGAC CGCAAGCGCTACACCAGCACCAAGGAGGTGCTGGACGCCACCCTGATC CACCAGAGCATCACCGGTCTGTACGAGACCCGCATCGACCTGAGCCAG CTGGGCGGCGACGGCGGCTCCGGACCTCCAAAGAAAAAGAGAAAAGTA TACCCCTACGACGTGCCCGACTACGCCTAATAACTCGAGCATGCATCT AGAGGGCCCTATTCTATAGTGTCACCTAAATGCTAGAGCTCGCTGATC AGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTC CCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTC CTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTC TATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGA AGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGA GGCGGAAAGAACCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGA GGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAG GCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCG TTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAA AATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGA TACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGC GTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAG GTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTA AGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGC AGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCT AACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTG AAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAA CAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATT ACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACG GGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAA TGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGAC AGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTA TTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACG ATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGA GACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCC GGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATC CAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTT AATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCA CGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCA AGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCA CTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCC GTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGA GAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGG GATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGA AAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGA TCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCT TTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAAT GCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATA CTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTC ATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGG GTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

Experimental Example 1: Selection of BMPRIA or BMPRII Gene Knock Out CHO Cell Clone

<1-1>Selection of BMPRIA Knock Out Clone

[0080] To select BMPRIA gene knock out clones, DNA was extracted from the CHO host cells prepared in Example 1, followed by PCR.

[0081] Particularly, the vector comprising gene scissors constructed in Example 1 was introduced into the CHO host cells. 2 days later, the CHO cells were inoculated in a 96 well plate at the density of 0.3 cells/well, followed by culture for 10-14 days. At this time, IMDM (Iscov's Modified Dulbecco's Medium) supplemented with 7% (v/v) dFBS (dialyzed fetal bovine serum) and hypoxanthine/thymidine (HT) was used as the medium. From the cultured cells, gDNA was extracted, followed by PCR to confirm that BMPRIA gene was knocked out. PCR was performed using the extracted gDNA as a template with the primers listed in Table 2 and Primestar (Takara) products according to the conditions shown in Table 3 below. Gene sequence of the PCR product was confirmed by DNA Sanger sequencing.

[0082] As a result, 6 BMPRIA knock out clones were selected. Particularly, compared with the wild type, clones #22, #35, #43, #47, #61 and #68 were confirmed to have deletions or additions of one or more nucleotide sequences in the 2.sup.nd exon of BMPRIA gene (FIG. 2 and Table 4). In FIG. 2, blue color indicates the 2.sup.nd exon sequence of BMPRIA gene which is the target to be knocked-out, green color indicates PAM nucleotide sequence and red color indicates deletions or additions of nucleotide sequences from the target gene.

TABLE-US-00002 TABLE2 Targetgene Primer Sequence(5.fwdarw.3) BMPRIAgene Forward GGAACTCACTCTGTAGAAGAGG 2.sup.ndexon primer (SEQ.ID.NO:14) Reverse GTGTTGGGGCACCCTTTGATC primer (SEQ.ID.NO:15)

TABLE-US-00003 TABLE 3 Temperature Time Cycle 95 C. 2 min 1 95 C. 30 sec 10 65 C. 1 min (1 C./cycle) 72 C. 1 min 30 sec 95 C. 30 sec 30 55 C. 1 min 72 C. 1 min 30 sec 72 C. 10 min

TABLE-US-00004 TABLE4 SEQ. BMPRIA ID. KO Nucleotidesequence NO: clone# (5-3) SEQ. #22 17 AGTAACACTTTAAGAAAGGCTAAAGTCACTCCATT ID. NO: 24 SEQ. 4 AGTAACACTTTAAGAAAGGCAAAGTTCCGGGGCTA ID. AAGTCACTCCATT NO: 25 SEQ. +1 AGTAACACTTTAAGAAAGGCAAAGTATCCTTCCGG ID. GGCTAAAGTCACTCCATT NO: 26 SEQ. #35 2 AGTAACACTTTAAGAAAGGCAAAGTATTCCGGGGC ID. TAAAGTCACTCCATT NO: 27 SEQ. 4 AGTAACACTTTAAGAAAGGCAAAGTACCGGGGCTA ID. AAGTCACTCCATT NO: 28 SEQ. +1 AGTAACACTTTAAGAAAGGCAAAGTATCCTTCCGG ID. GGCTAAAGTCACTCCATT NO: 29 SEQ. +2 AGTAACACTTTAAGAAAGGCAAAGTATCCTTTCCG ID. GGGCTAAAGTCACTCCATT NO: 30 SEQ. #43 +1 AGTAACACTTTAAGAAAGGCAAAGTATCCTTCCGG ID. GGCTAAAGTCACTCCATT NO: 31 SEQ. #47 35 AGTAAAGTCACTCCATT ID. NO: 32 SEQ. 3, AGTAACACTTTAAGAAAGGCAAAGTATACCGGGGC ID. +1 TAAAGTCACTCCATT NO: 33 SEQ. #61 +115 AGTAACACTTTAAGAAAGGCAAAGTATCCTCAGAC ID. CATCCTGGACTTCCTGAAGAGCGACGGCTTCGCCA NO: ACCGCAACTTCATGCAGCTGATCCACGACGACAGC 34 CTGACCTTCAAGGAGGACATCCAGAAGGCCCAGGT GAGCGTCGGGGCTAAAGTCACTCCATT SEQ. #68 +49 AGTAACACTTTAAGAAAGGCAAAGTATCCTCTGGT ID. CTTGGAACACTTCCAAATCTCAGTGAAAAGCGAAG NO: CCAGTGTTTCCGGGGCTAAAGTCACTCCATT 35

<1-2>Selection of BMPRII Knock Out Clone

[0083] To select BMPRII gene knock out clones, DNA was extracted from the CHO host cells prepared in Example 1, followed by PCR.

[0084] Particularly, the host cells prepared in Example 2 were cultured and gDNA was extracted by the same manner as described in Experimental Example <1-1>. To confirm the knockout clones, junction PCR and out-out PCR were performed. Junction PCR is a kind of PCR performed with the primers targeting the non-knocked-out region and the knock-out region. In this PCR, when a gene is knocked-out, PCR product does not appear on gel. Out-out PCR is a kind of PCR performed with the primers targeting both sides of the knocked-out region. In this PCR, when a gene is knocked-out, a smaller sized PCR product appears on gel, compared with when a gene is not knocked-out. Primers used for the junction PCR and out-out PCR above are shown in Table 5 below. As a result, 3 BMPRII gene knock out clones were selected. Particularly, junction PCR product and out-out PCR product were electrophoresed. As a result, compared with the wild type, bands of clones #20, #52 and #118 were not observed on junction PCR gel, while the bands were observed on out-out PCR gel (Table 3).

TABLE-US-00005 TABLE5 Target PCR gene Primer Sequence(5.fwdarw.3) Junction 2.sup.nd-3.sup.rd Forward AGAACGGCTGTGTGCATTTA PCR exons primer (SEQ.ID.NO:16) ofBMPRII Reverse CCAATGTGAGACCAACATCCT gene primer (SEQ.ID.NO:17) 5.sup.th-6.sup.th Forward CTGCTGCAGCTCTTCTTAACT exons primer (SEQ.ID.NO:18) ofBMPRII Reverse CGCTCATCCAAGGAACCTTTA gene primer (SEQ.ID.NO:19) Out-out Genome Forward CTAACTCACAGAGATTGCCTACC PCR between primer (SEQ.ID.NO:20) 1.sup.stand2.sup.nd Reverse CCCAGCTCCTCCACTTTATTT exon primer (SEQ.ID.NO:21) ofBMPRII geneand genome between 5.sup.thand6.sup.th exon ofBMPRII gene

Experimental Example 2: Target Protein Production in BMPRIA or BMPRII Gene Knock Out CHO Cell Line

[0085] To evaluate the productivity of a target protein in the BMPRIA or BMPRII gene knock out CHO host cell line selected in Experimental Example <1-2>, the target protein rhBMP4 (recombinant human BMP4) expression was induced in each CHO cell line, and then compared with that of the wild type CHO cell line.

[0086] First, the wild type CHO host cell line, the BMPRIA knock out CHO host cell lines and the BMPRII knock out CHO host cell lines selected in Experimental Example 1 were cultured in IMDM (Iscov's Modified Dulbecco's Medium) supplemented with 7%(v/v) dFBS (dialyzed fetal bovine serum) and hypoxanthine/thymidine (HT) via adhesion culture. A vector containing genes encoding dhfr (dihydrofolate reductase, Table 6, SEQ. ID. NO: 23) and rhBMP4 (Table 6, SEQ. ID. NO: 22) was introduced into the cultured CHO cells by using lipofectamine (lipofectamine 2000, Life technology). The vector was constructed by using pOptiVEC-TOPO vector cloning kit (ThermoFisher Scientific, Catalog number: 12744017) and the gene was introduced by using lipofectamine (lipofectamine 2000, Life technology). To induce the expression of the recombinant protein rhBMP4 gene in the cells introduced with the vector comprising the gene encoding dhfr and rhBMP4 using dhfr/MTX system, the process of amplifying the productivity of the target protein rhBMP4 was repeated while increasing the concentration of MTX (1 nM10 nM100 nM1 M). Finally, the pool of viable cells in which the expression of the rhBMP4 gene was amplified at 1 M MTX was prepared. To select clones from the prepared cell pool, cells were inoculated in a 96 well plate at the density of 0.3 cells/well, followed by culture for 10-14 days. After the culture, the cells that were grown normally were selected and the productivity of rhBMP4 of the selected clones was confirmed by ELISA.

[0087] As a result, clone #47 (IA-KO-47) selected from the BMPRIA gene knock out clones, and clones #20 and #118 (II-KO-20, II-KO-118) selected from the BMPRII gene knock clones demonstrated higher rhBMP4 productivity at average than that of the wile type clone (FIG. 4).

TABLE-US-00006 TABLE6 SEQ. ID. Pro- NO: tein Aminoacidsequence SEQ. rhEMP4 MIPGNRMLMVVLLCQVLLGGASHASLIPETGKKKVAEI ID. QGHAGGRRSGQSHELLRDFEATLLQMFGLRRRPQPSKS NO: AVIPDYMRDLYRLQSGEEEEEQIHSTGLEYPERPASRA 22 NTVRSFHHEEHLENIPGTSENSAFRFLFNLSSIPENEV ISSAELRLFREQVDQGPDWERGFHRINIYEVMKPPAEV VPGHLITRLLDTRLVHHNVTRWETFDVSPAVLRWTREK QPNYGLAIEVTHLHQTRTHQGQHVRISRSLPQGSGNWA QLRPLLVTFGHDGRGHALTRRRRAKRSPKHHSQRARKK NKNCRRHSLYVDFSDVGWNDWIVAPPGYQAFYCHGDCP FPLADHLNSTNHAIVQTLVNSVNSSIPKACCVPTELSA ISMLYLDEYDKVVLKNYQEMVVEGCGCR SEQ. DHFR ATGGTTCGACCATTGAACTGCATCGTCGCCGTGTCCCA ID. AAATATGGGGATTGGCAAGAACGGAGACCTACCCTGGC NO: CTCCGCTCAGGAACGAGTTCAAGTACTTCCAAAGAATG 23 ACCACAACCTCTTCAGTGGAAGGTAAACAGAATCTGGT GATTATGGGTAGGAAAACCTGGTTCTCCATTCCTGAGA AGAATCGACCTTTAAAGGACAGAATTAATATAGTTCTC AGTAGAGAACTCAAAGAACCACCACGAGGAGCTCATTT TCTTGCCAAAAGTTTGGATGATGCCTTAAGACTTATTG AACAACCGGAATTGGCAAGTAAAGTAGACATGGTTTGG ATAGTCGGAGGCAGTTCTGTTTACCAGGAAGCCATGAA TCAACCAGGCCACCTCAGACTCTTTGTGACAAGGATCA TGCAGGAATTTGAAAGTGACACGTTTTTCCCAGAAATT GATTTGGGGAAATATAAACTTCTCCCAGAATACCCAGG CGTCCTCTCTGAGGTCCAGGAGGAAAAAGGCATCAAGT ATAAGTTTGAAGTCTACGAGAAGAAAGACTAA

Experimental Example 3: Maximum Production of Target Protein in BMPRIA or BMPRII Gene Knock Out CHO Cell Line

[0088] One BMPRIA gene knock out clone (IA-KO-47) and two BMPRII gene knock out clones (II-KO-20, II-KO-118) demonstrating higher target protein productivity than that of the wild type in Experimental Example 2 were selected. The three knock out clones selected above were batch-cultured by the same manner as described in Experimental Example 2 via adhesion culture. The culture medium of each cell line was collected daily and the concentration of rhBMP4 was measured by ELISA. Then, the rhBMP4 concentration at the highest productivity was compared with that of the wild type. Five clones showing high target protein productivity were selected and used as the wild type. The maximum production of the 5 wild type clones and the 3 knock out clones were averaged.

[0089] As a result, it was confirmed that the maximum productivity of BMPRIA or BMPRII knock out CHO cell line (KO) was significantly higher than that of the wild type (DG44wt) (FIG. 5).

Experimental Example 4: Target Protein Production in Suspension Culture of BMPRIA or BMPRII Gene Knock Out CHO Cell Line

[0090] Protein productivity was evaluated by the same manner as described in Experimental Example 3 except that clones were suspension-cultured at this time. The culture process was as follows. First, each clone was inoculated in 30 ml of CD-OptiCHO medium (Invitrogen, Burlington, USA) supplemented with 1 M MTX and 8 mM glutamine at the density of 5.010.sup.5 cells/ml, followed by culture at 110 rpm at 37 C. From the third day after the start of the culture, fed-batch culture was performed with adding 1%(v/v) Cell boost 2, 5, 6 supplement (HyClone) daily. During the culture, 1 ml of the medium was collected every day to measure the cell concentration, and the concentration of rhBMP4 was measured by ELISA.

[0091] As a result, the accumulated cell concentration of BMPRIA or BMPRII gene knock out CHO cell line (KO) was higher than that of the wild type (DG44wt), indicating that the signal transduction was interrupted by knocking out the BMP receptor, so that the cell growth was successfully improved (FIG. 6). As a result of the measurement of the rhBMP4 concentration, the maximum productivity of the BMPRIA or BMPRII knock out CHO cell line (KO) was remarkably higher than that of the wild type (DG44wt) (FIG. 7).