CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice
Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder...
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| Veröffentlicht in: | Scientific reports 2016-08, Vol.6 (1), p.31666, Article 31666 |
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| creator | Oji, Asami Noda, Taichi Fujihara, Yoshitaka Miyata, Haruhiko Kim, Yeon Joo Muto, Masanaga Nozawa, Kaori Matsumura, Takafumi Isotani, Ayako Ikawa, Masahito |
| description | Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene,
Dnajb13
, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that
Dnajb13
is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism. |
| doi_str_mv | 10.1038/srep31666 |
| format | Article |
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Dnajb13
, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that
Dnajb13
is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.</description><identifier>ISSN: 2045-2322</identifier><identifier>EISSN: 2045-2322</identifier><identifier>DOI: 10.1038/srep31666</identifier><identifier>PMID: 27530713</identifier><language>eng</language><publisher>London: Nature Publishing Group UK</publisher><subject>38/109 ; 631/1647 ; 631/61 ; 96/100 ; Animals ; Apoptosis Regulatory Proteins ; Chimera - genetics ; Cilia ; CRISPR ; CRISPR-Cas Systems ; Editing ; Embryo cells ; Embryonic Stem Cells - metabolism ; Female ; Gene Deletion ; Gene Editing - methods ; Gene Knock-In Techniques ; Genes, Lethal ; Genome editing ; Genomes ; HSP40 Heat-Shock Proteins - genetics ; Humanities and Social Sciences ; Hydrocephalus ; Hydrocephalus - genetics ; Hydrocephalus - pathology ; INDEL Mutation ; Male ; Mice ; Mice, Inbred C57BL ; Mice, Inbred DBA ; Mice, Inbred ICR ; Mice, Transgenic ; Molecular Chaperones ; Mosaicism ; multidisciplinary ; Mutation ; Point Mutation ; Pregnancy ; Ribonucleic acid ; RNA ; RNA, Guide, CRISPR-Cas Systems - genetics ; Science ; Science (multidisciplinary) ; Sperm Motility - genetics ; Spermatozoa - abnormalities ; Stem cell transplantation ; Stem cells</subject><ispartof>Scientific reports, 2016-08, Vol.6 (1), p.31666, Article 31666</ispartof><rights>The Author(s) 2016</rights><rights>Copyright Nature Publishing Group Aug 2016</rights><rights>Copyright © 2016, The Author(s) 2016 The Author(s)</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c548t-c06115d4981b5f84aba9d843eabeead82a7afea91a4806e3648f9ef19f84d6663</citedby><cites>FETCH-LOGICAL-c548t-c06115d4981b5f84aba9d843eabeead82a7afea91a4806e3648f9ef19f84d6663</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987700/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987700/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,27901,27902,41096,42165,51551,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/27530713$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Oji, Asami</creatorcontrib><creatorcontrib>Noda, Taichi</creatorcontrib><creatorcontrib>Fujihara, Yoshitaka</creatorcontrib><creatorcontrib>Miyata, Haruhiko</creatorcontrib><creatorcontrib>Kim, Yeon Joo</creatorcontrib><creatorcontrib>Muto, Masanaga</creatorcontrib><creatorcontrib>Nozawa, Kaori</creatorcontrib><creatorcontrib>Matsumura, Takafumi</creatorcontrib><creatorcontrib>Isotani, Ayako</creatorcontrib><creatorcontrib>Ikawa, Masahito</creatorcontrib><title>CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice</title><title>Scientific reports</title><addtitle>Sci Rep</addtitle><addtitle>Sci Rep</addtitle><description>Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene,
Dnajb13
, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that
Dnajb13
is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.</description><subject>38/109</subject><subject>631/1647</subject><subject>631/61</subject><subject>96/100</subject><subject>Animals</subject><subject>Apoptosis Regulatory Proteins</subject><subject>Chimera - genetics</subject><subject>Cilia</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Editing</subject><subject>Embryo cells</subject><subject>Embryonic Stem Cells - metabolism</subject><subject>Female</subject><subject>Gene Deletion</subject><subject>Gene Editing - methods</subject><subject>Gene Knock-In Techniques</subject><subject>Genes, Lethal</subject><subject>Genome editing</subject><subject>Genomes</subject><subject>HSP40 Heat-Shock Proteins - genetics</subject><subject>Humanities and Social Sciences</subject><subject>Hydrocephalus</subject><subject>Hydrocephalus - genetics</subject><subject>Hydrocephalus - pathology</subject><subject>INDEL Mutation</subject><subject>Male</subject><subject>Mice</subject><subject>Mice, Inbred C57BL</subject><subject>Mice, Inbred DBA</subject><subject>Mice, Inbred ICR</subject><subject>Mice, Transgenic</subject><subject>Molecular Chaperones</subject><subject>Mosaicism</subject><subject>multidisciplinary</subject><subject>Mutation</subject><subject>Point Mutation</subject><subject>Pregnancy</subject><subject>Ribonucleic acid</subject><subject>RNA</subject><subject>RNA, Guide, CRISPR-Cas Systems - genetics</subject><subject>Science</subject><subject>Science (multidisciplinary)</subject><subject>Sperm Motility - genetics</subject><subject>Spermatozoa - abnormalities</subject><subject>Stem cell transplantation</subject><subject>Stem cells</subject><issn>2045-2322</issn><issn>2045-2322</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2016</creationdate><recordtype>article</recordtype><sourceid>C6C</sourceid><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNplkU1LAzEQhoMoKurBPyABTwq1yWY_kosgpWpBUFo9h9ns7BrZL5Ot4L83pbVUzGUyzJP3zcwQcs7ZDWdCjr3DXvA0TffIccTiZBSJKNrfuR-RM-8_WDhJpGKuDslRlCWCZVwcE5jMZ4uX-XgCXtEGCwsDFrTCtmuQhnSwbUVtS6cLarCuPYW2oHYIse9ra2CwXUvLzlHzbht01gQA6m9v_epVYw2ekoMSao9nm3hC3u6nr5PH0dPzw2xy9zQySSyHkWEp50kRK8nzpJQx5KAKGQuEHBEKGUEGJYLiEEuWokhjWSosuQpsEboXJ-R2rdsv89CIwXZwUOve2Qbct-7A6r-V1r7rqvvSwTLLGAsClxsB130u0Q_6o1u60I3XXDEhJE-FDNTVmjKu82H25daBM71aiN4uJLAXu1_akr_jD8D1GvCh1Fbodiz_qf0AFm6VWg</recordid><startdate>20160817</startdate><enddate>20160817</enddate><creator>Oji, Asami</creator><creator>Noda, Taichi</creator><creator>Fujihara, Yoshitaka</creator><creator>Miyata, Haruhiko</creator><creator>Kim, Yeon Joo</creator><creator>Muto, Masanaga</creator><creator>Nozawa, Kaori</creator><creator>Matsumura, Takafumi</creator><creator>Isotani, Ayako</creator><creator>Ikawa, Masahito</creator><general>Nature Publishing Group UK</general><general>Nature Publishing Group</general><scope>C6C</scope><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>3V.</scope><scope>7X7</scope><scope>7XB</scope><scope>88A</scope><scope>88E</scope><scope>88I</scope><scope>8FE</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>LK8</scope><scope>M0S</scope><scope>M1P</scope><scope>M2P</scope><scope>M7P</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>Q9U</scope><scope>5PM</scope></search><sort><creationdate>20160817</creationdate><title>CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice</title><author>Oji, Asami ; Noda, Taichi ; Fujihara, Yoshitaka ; Miyata, Haruhiko ; Kim, Yeon Joo ; Muto, Masanaga ; Nozawa, Kaori ; Matsumura, Takafumi ; Isotani, Ayako ; Ikawa, Masahito</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c548t-c06115d4981b5f84aba9d843eabeead82a7afea91a4806e3648f9ef19f84d6663</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2016</creationdate><topic>38/109</topic><topic>631/1647</topic><topic>631/61</topic><topic>96/100</topic><topic>Animals</topic><topic>Apoptosis Regulatory Proteins</topic><topic>Chimera - genetics</topic><topic>Cilia</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Editing</topic><topic>Embryo cells</topic><topic>Embryonic Stem Cells - metabolism</topic><topic>Female</topic><topic>Gene Deletion</topic><topic>Gene Editing - methods</topic><topic>Gene Knock-In Techniques</topic><topic>Genes, Lethal</topic><topic>Genome editing</topic><topic>Genomes</topic><topic>HSP40 Heat-Shock Proteins - genetics</topic><topic>Humanities and Social Sciences</topic><topic>Hydrocephalus</topic><topic>Hydrocephalus - genetics</topic><topic>Hydrocephalus - pathology</topic><topic>INDEL Mutation</topic><topic>Male</topic><topic>Mice</topic><topic>Mice, Inbred C57BL</topic><topic>Mice, Inbred DBA</topic><topic>Mice, Inbred ICR</topic><topic>Mice, Transgenic</topic><topic>Molecular Chaperones</topic><topic>Mosaicism</topic><topic>multidisciplinary</topic><topic>Mutation</topic><topic>Point Mutation</topic><topic>Pregnancy</topic><topic>Ribonucleic acid</topic><topic>RNA</topic><topic>RNA, Guide, CRISPR-Cas Systems - genetics</topic><topic>Science</topic><topic>Science (multidisciplinary)</topic><topic>Sperm Motility - genetics</topic><topic>Spermatozoa - abnormalities</topic><topic>Stem cell transplantation</topic><topic>Stem cells</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Oji, Asami</creatorcontrib><creatorcontrib>Noda, Taichi</creatorcontrib><creatorcontrib>Fujihara, Yoshitaka</creatorcontrib><creatorcontrib>Miyata, Haruhiko</creatorcontrib><creatorcontrib>Kim, Yeon Joo</creatorcontrib><creatorcontrib>Muto, Masanaga</creatorcontrib><creatorcontrib>Nozawa, Kaori</creatorcontrib><creatorcontrib>Matsumura, Takafumi</creatorcontrib><creatorcontrib>Isotani, Ayako</creatorcontrib><creatorcontrib>Ikawa, Masahito</creatorcontrib><collection>Springer Nature OA Free Journals</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Biology Database (Alumni Edition)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Science Database (Alumni Edition)</collection><collection>ProQuest SciTech Collection</collection><collection>ProQuest Natural Science Collection</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest One Sustainability</collection><collection>ProQuest Central UK/Ireland</collection><collection>ProQuest Central Essentials</collection><collection>Biological Science Collection</collection><collection>ProQuest Central</collection><collection>Natural Science Collection</collection><collection>ProQuest One Community College</collection><collection>ProQuest Central Korea</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Central Student</collection><collection>SciTech Premium Collection</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>ProQuest Biological Science Collection</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Science Database</collection><collection>Biological Science Database</collection><collection>Publicly Available Content Database</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central Basic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Scientific reports</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Oji, Asami</au><au>Noda, Taichi</au><au>Fujihara, Yoshitaka</au><au>Miyata, Haruhiko</au><au>Kim, Yeon Joo</au><au>Muto, Masanaga</au><au>Nozawa, Kaori</au><au>Matsumura, Takafumi</au><au>Isotani, Ayako</au><au>Ikawa, Masahito</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice</atitle><jtitle>Scientific reports</jtitle><stitle>Sci Rep</stitle><addtitle>Sci Rep</addtitle><date>2016-08-17</date><risdate>2016</risdate><volume>6</volume><issue>1</issue><spage>31666</spage><pages>31666-</pages><artnum>31666</artnum><issn>2045-2322</issn><eissn>2045-2322</eissn><abstract>Targeted gene disrupted mice can be efficiently generated by expressing a single guide RNA (sgRNA)/CAS9 complex in the zygote. However, the limited success of complicated genome editing, such as large deletions, point mutations, and knockins, remains to be improved. Further, the mosaicism in founder generations complicates the genotypic and phenotypic analyses in these animals. Here we show that large deletions with two sgRNAs as well as dsDNA-mediated point mutations are efficient in mouse embryonic stem cells (ESCs). The dsDNA-mediated gene knockins are also feasible in ESCs. Finally, we generated chimeric mice with biallelic mutant ESCs for a lethal gene,
Dnajb13
, and analyzed their phenotypes. Not only was the lethal phenotype of hydrocephalus suppressed, but we also found that
Dnajb13
is required for sperm cilia formation. The combination of biallelic genome editing in ESCs and subsequent chimeric analysis provides a useful tool for rapid gene function analysis in the whole organism.</abstract><cop>London</cop><pub>Nature Publishing Group UK</pub><pmid>27530713</pmid><doi>10.1038/srep31666</doi><oa>free_for_read</oa></addata></record> |
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| subjects | 38/109 631/1647 631/61 96/100 Animals Apoptosis Regulatory Proteins Chimera - genetics Cilia CRISPR CRISPR-Cas Systems Editing Embryo cells Embryonic Stem Cells - metabolism Female Gene Deletion Gene Editing - methods Gene Knock-In Techniques Genes, Lethal Genome editing Genomes HSP40 Heat-Shock Proteins - genetics Humanities and Social Sciences Hydrocephalus Hydrocephalus - genetics Hydrocephalus - pathology INDEL Mutation Male Mice Mice, Inbred C57BL Mice, Inbred DBA Mice, Inbred ICR Mice, Transgenic Molecular Chaperones Mosaicism multidisciplinary Mutation Point Mutation Pregnancy Ribonucleic acid RNA RNA, Guide, CRISPR-Cas Systems - genetics Science Science (multidisciplinary) Sperm Motility - genetics Spermatozoa - abnormalities Stem cell transplantation Stem cells |
| title | CRISPR/Cas9 mediated genome editing in ES cells and its application for chimeric analysis in mice |
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