Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems
Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transge...
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| Veröffentlicht in: | Journal of bioscience and bioengineering 2018-05, Vol.125 (5), p.599-605 |
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| container_title | Journal of bioscience and bioengineering |
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| creator | Kawabe, Yoshinori Komatsu, Shinya Komatsu, Shodai Murakami, Mai Ito, Akira Sakuma, Tetsushi Nakamura, Takahiro Yamamoto, Takashi Kamihira, Masamichi |
| description | Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering. |
| doi_str_mv | 10.1016/j.jbiosc.2017.12.003 |
| format | Article |
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For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.</description><identifier>ISSN: 1389-1723</identifier><identifier>EISSN: 1347-4421</identifier><identifier>DOI: 10.1016/j.jbiosc.2017.12.003</identifier><identifier>PMID: 29295784</identifier><language>eng</language><publisher>Japan: Elsevier B.V</publisher><subject>Animals ; CHO Cells ; Cricetinae ; Cricetulus ; CRIS-PITCh ; CRISPR-Cas Systems ; CRISPR/Cas9 ; Gene Knock-In Techniques - methods ; Genes, Immunoglobulin ; Genetic Loci ; Hypoxanthine Phosphoribosyltransferase - genetics ; Immunoglobulin Fc Fragments - genetics ; Mutagenesis, Insertional - methods ; scFv-Fc ; Single-Chain Antibodies - genetics ; Targeted gene knock-in ; Transgenes</subject><ispartof>Journal of bioscience and bioengineering, 2018-05, Vol.125 (5), p.599-605</ispartof><rights>2017 The Society for Biotechnology, Japan</rights><rights>Copyright © 2017 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c489t-d8bf39b570ff9270faac500fba9c6301e8211c622d768e957333c1d61da785313</citedby><cites>FETCH-LOGICAL-c489t-d8bf39b570ff9270faac500fba9c6301e8211c622d768e957333c1d61da785313</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1389172317310691$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,776,780,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/29295784$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Kawabe, Yoshinori</creatorcontrib><creatorcontrib>Komatsu, Shinya</creatorcontrib><creatorcontrib>Komatsu, Shodai</creatorcontrib><creatorcontrib>Murakami, Mai</creatorcontrib><creatorcontrib>Ito, Akira</creatorcontrib><creatorcontrib>Sakuma, Tetsushi</creatorcontrib><creatorcontrib>Nakamura, Takahiro</creatorcontrib><creatorcontrib>Yamamoto, Takashi</creatorcontrib><creatorcontrib>Kamihira, Masamichi</creatorcontrib><title>Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems</title><title>Journal of bioscience and bioengineering</title><addtitle>J Biosci Bioeng</addtitle><description>Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.</description><subject>Animals</subject><subject>CHO Cells</subject><subject>Cricetinae</subject><subject>Cricetulus</subject><subject>CRIS-PITCh</subject><subject>CRISPR-Cas Systems</subject><subject>CRISPR/Cas9</subject><subject>Gene Knock-In Techniques - methods</subject><subject>Genes, Immunoglobulin</subject><subject>Genetic Loci</subject><subject>Hypoxanthine Phosphoribosyltransferase - genetics</subject><subject>Immunoglobulin Fc Fragments - genetics</subject><subject>Mutagenesis, Insertional - methods</subject><subject>scFv-Fc</subject><subject>Single-Chain Antibodies - genetics</subject><subject>Targeted gene knock-in</subject><subject>Transgenes</subject><issn>1389-1723</issn><issn>1347-4421</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kc2O0zAUhSMEYoaBN0DISzbJ-CeJ4w0SiqZQaSRGQ1lbjn3TutPExTep1EfgrXHowJKNfW19514fnyx7z2jBKKtv98W-8wFtwSmTBeMFpeJFds1EKfOy5OzlUjcqZ5KLq-wN4p4mkEr2OrviiqtKNuV19mtj4hYmcORpDPYp9yMJPTEjQbs65Subysl3wZ3JFkYgfpwCmXZAdsc4kUOwMy58u_MjYLo1A04QSTiZeCYWDgckM_pxS9rH9feHx9vWoEot3Z9z_rDetDuC56QZ8G32qjcHhHfP-032Y3W3ab_m99--rNvP97ktGzXlrul6obpK0r5XPK3G2IrSvjPK1oIyaDhjtubcybqB5FIIYZmrmTOyqQQTN9nHS99jDD9nwEkPHpenmhHCjJqppuSVkqxOaHlBbQyIEXp9jH5I1jSjeglB7_UlBL2EoBnXKYQk-_A8Ye4GcP9Ef389AZ8uACSfJw9Ro_UwWnA-gp20C_7_E34Dm3OZTA</recordid><startdate>20180501</startdate><enddate>20180501</enddate><creator>Kawabe, Yoshinori</creator><creator>Komatsu, Shinya</creator><creator>Komatsu, Shodai</creator><creator>Murakami, Mai</creator><creator>Ito, Akira</creator><creator>Sakuma, Tetsushi</creator><creator>Nakamura, Takahiro</creator><creator>Yamamoto, Takashi</creator><creator>Kamihira, Masamichi</creator><general>Elsevier B.V</general><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>20180501</creationdate><title>Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems</title><author>Kawabe, Yoshinori ; Komatsu, Shinya ; Komatsu, Shodai ; Murakami, Mai ; Ito, Akira ; Sakuma, Tetsushi ; Nakamura, Takahiro ; Yamamoto, Takashi ; Kamihira, Masamichi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c489t-d8bf39b570ff9270faac500fba9c6301e8211c622d768e957333c1d61da785313</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Animals</topic><topic>CHO Cells</topic><topic>Cricetinae</topic><topic>Cricetulus</topic><topic>CRIS-PITCh</topic><topic>CRISPR-Cas Systems</topic><topic>CRISPR/Cas9</topic><topic>Gene Knock-In Techniques - methods</topic><topic>Genes, Immunoglobulin</topic><topic>Genetic Loci</topic><topic>Hypoxanthine Phosphoribosyltransferase - genetics</topic><topic>Immunoglobulin Fc Fragments - genetics</topic><topic>Mutagenesis, Insertional - methods</topic><topic>scFv-Fc</topic><topic>Single-Chain Antibodies - genetics</topic><topic>Targeted gene knock-in</topic><topic>Transgenes</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Kawabe, Yoshinori</creatorcontrib><creatorcontrib>Komatsu, Shinya</creatorcontrib><creatorcontrib>Komatsu, Shodai</creatorcontrib><creatorcontrib>Murakami, Mai</creatorcontrib><creatorcontrib>Ito, Akira</creatorcontrib><creatorcontrib>Sakuma, Tetsushi</creatorcontrib><creatorcontrib>Nakamura, Takahiro</creatorcontrib><creatorcontrib>Yamamoto, Takashi</creatorcontrib><creatorcontrib>Kamihira, Masamichi</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Journal of bioscience and bioengineering</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Kawabe, Yoshinori</au><au>Komatsu, Shinya</au><au>Komatsu, Shodai</au><au>Murakami, Mai</au><au>Ito, Akira</au><au>Sakuma, Tetsushi</au><au>Nakamura, Takahiro</au><au>Yamamoto, Takashi</au><au>Kamihira, Masamichi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems</atitle><jtitle>Journal of bioscience and bioengineering</jtitle><addtitle>J Biosci Bioeng</addtitle><date>2018-05-01</date><risdate>2018</risdate><volume>125</volume><issue>5</issue><spage>599</spage><epage>605</epage><pages>599-605</pages><issn>1389-1723</issn><eissn>1347-4421</eissn><abstract>Chinese hamster ovary (CHO) cells have been used as host cells for the production of pharmaceutical proteins. For the high and stable production of target proteins, the transgene should be integrated into a suitable genomic locus of host cells. Here, we generated knock-in CHO cells, in which transgene cassettes without a vector backbone sequence were integrated into the hprt locus of the CHO genome using clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 and CRISPR-mediated precise integration into target chromosome (CRIS-PITCh) systems. We investigated the efficiency of targeted knock-in of transgenes using these systems. As a practical example, we generated knock-in CHO cells producing an scFv-Fc antibody using the CRIS-PITCh system mediated by microhomology sequences for targeting. We found that the CRIS-PITCh system can facilitate targeted knock-in for CHO cell engineering.</abstract><cop>Japan</cop><pub>Elsevier B.V</pub><pmid>29295784</pmid><doi>10.1016/j.jbiosc.2017.12.003</doi><tpages>7</tpages></addata></record> |
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| ispartof | Journal of bioscience and bioengineering, 2018-05, Vol.125 (5), p.599-605 |
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| language | eng |
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| source | MEDLINE; Elsevier ScienceDirect Journals |
| subjects | Animals CHO Cells Cricetinae Cricetulus CRIS-PITCh CRISPR-Cas Systems CRISPR/Cas9 Gene Knock-In Techniques - methods Genes, Immunoglobulin Genetic Loci Hypoxanthine Phosphoribosyltransferase - genetics Immunoglobulin Fc Fragments - genetics Mutagenesis, Insertional - methods scFv-Fc Single-Chain Antibodies - genetics Targeted gene knock-in Transgenes |
| title | Targeted knock-in of an scFv-Fc antibody gene into the hprt locus of Chinese hamster ovary cells using CRISPR/Cas9 and CRIS-PITCh systems |
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