Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis
Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletio...
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| creator | Ipsaro, Jonathan J Shen, Chen Arai, Eri Xu, Yali Kinney, Justin B Joshua-Tor, Leemor Vakoc, Christopher R Shi, Junwei |
| description | Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development. |
| doi_str_mv | 10.1371/journal.pone.0172177 |
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Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0172177</identifier><identifier>PMID: 28231254</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>Alleles ; Animals ; Antineoplastic Agents - pharmacology ; Benzimidazoles - pharmacology ; Biology ; Biology and Life Sciences ; Cancer ; Cell Line ; Cell Line, Tumor ; Cell Proliferation - drug effects ; Clonal deletion ; Cloning ; Clustered Regularly Interspaced Short Palindromic Repeats ; CRISPR ; CRISPR-Cas Systems ; Deoxyribonucleic acid ; Developmental stages ; Discovery tools ; DNA ; DNA methylation ; Drug development ; Drug resistance ; Drug Resistance, Neoplasm ; Engineering and Technology ; Enhancer of Zeste Homolog 2 Protein - genetics ; Enhancer of Zeste Homolog 2 Protein - metabolism ; Genes ; Genetic aspects ; Health aspects ; HEK293 Cells ; Humans ; INDEL Mutation ; Inhibitors ; Insertion ; Laboratories ; Leukemia ; Loci ; Lysine ; Medical research ; Methyltransferases - genetics ; Methyltransferases - metabolism ; Mice ; Models, Molecular ; Mutagenesis ; Mutation ; Neoplasms - drug therapy ; Neoplasms - genetics ; Neoplasms - metabolism ; Plasmids ; Proteins ; Research and Analysis Methods</subject><ispartof>PloS one, 2017-02, Vol.12 (2), p.e0172177</ispartof><rights>COPYRIGHT 2017 Public Library of Science</rights><rights>2017 Ipsaro et al. This is an open access article distributed under the terms of the Creative Commons Attribution License: http://creativecommons.org/licenses/by/4.0/ (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2017 Ipsaro et al 2017 Ipsaro et al</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c725t-3463d2c85135e727f723501b40e9140ec530936affae559fcb9dbca4ce6dbcf63</citedby><cites>FETCH-LOGICAL-c725t-3463d2c85135e727f723501b40e9140ec530936affae559fcb9dbca4ce6dbcf63</cites><orcidid>0000-0002-8427-6316</orcidid></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322889/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC5322889/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,725,778,782,862,883,2098,2917,23853,27911,27912,53778,53780,79357,79358</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28231254$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Wang, Tony T.</contributor><creatorcontrib>Ipsaro, Jonathan J</creatorcontrib><creatorcontrib>Shen, Chen</creatorcontrib><creatorcontrib>Arai, Eri</creatorcontrib><creatorcontrib>Xu, Yali</creatorcontrib><creatorcontrib>Kinney, Justin B</creatorcontrib><creatorcontrib>Joshua-Tor, Leemor</creatorcontrib><creatorcontrib>Vakoc, Christopher R</creatorcontrib><creatorcontrib>Shi, Junwei</creatorcontrib><title>Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>Genetic alterations conferring resistance to the effects of chemical inhibitors are valuable tools for validating on-target effects in cells. Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.</description><subject>Alleles</subject><subject>Animals</subject><subject>Antineoplastic Agents - pharmacology</subject><subject>Benzimidazoles - pharmacology</subject><subject>Biology</subject><subject>Biology and Life Sciences</subject><subject>Cancer</subject><subject>Cell Line</subject><subject>Cell Line, Tumor</subject><subject>Cell Proliferation - drug effects</subject><subject>Clonal deletion</subject><subject>Cloning</subject><subject>Clustered Regularly Interspaced Short Palindromic Repeats</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>Deoxyribonucleic acid</subject><subject>Developmental stages</subject><subject>Discovery tools</subject><subject>DNA</subject><subject>DNA methylation</subject><subject>Drug development</subject><subject>Drug resistance</subject><subject>Drug Resistance, Neoplasm</subject><subject>Engineering and Technology</subject><subject>Enhancer of Zeste Homolog 2 Protein - genetics</subject><subject>Enhancer of Zeste Homolog 2 Protein - metabolism</subject><subject>Genes</subject><subject>Genetic aspects</subject><subject>Health aspects</subject><subject>HEK293 Cells</subject><subject>Humans</subject><subject>INDEL Mutation</subject><subject>Inhibitors</subject><subject>Insertion</subject><subject>Laboratories</subject><subject>Leukemia</subject><subject>Loci</subject><subject>Lysine</subject><subject>Medical research</subject><subject>Methyltransferases - genetics</subject><subject>Methyltransferases - metabolism</subject><subject>Mice</subject><subject>Models, Molecular</subject><subject>Mutagenesis</subject><subject>Mutation</subject><subject>Neoplasms - drug therapy</subject><subject>Neoplasms - genetics</subject><subject>Neoplasms - metabolism</subject><subject>Plasmids</subject><subject>Proteins</subject><subject>Research and Analysis Methods</subject><issn>1932-6203</issn><issn>1932-6203</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>AZQEC</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><sourceid>DWQXO</sourceid><sourceid>GNUQQ</sourceid><sourceid>DOA</sourceid><recordid>eNqNk12L1DAUhoso7jr6D0QLguhFx3w0SXsjLIMfAwsrs-ptSNPTTpZMMiat6L83dWaXqeyFFJqSPu-bk_ORZc8xWmIq8LsbPwan7HLvHSwRFgQL8SA7xzUlBSeIPjz5PsuexHiDEKMV54-zM1IRigkrz7Nmo_amzXtwENRgvMt9l7dh7IsA0cRBOQ25shYsxFwNObjWJ9iPMbdem3yMxvX5arO-_rIpVirWuXEt2Hw3DmoyTR5Ps0edshGeHddF9u3jh6-rz8Xl1af16uKy0IKwoaAlpy3RFcOUgSCiE4QyhJsSQY3TSzOKaspV1ylgrO50U7eNVqUGntaO00X28uC7tz7KY3qixJXAjHCUxItsfSBar27kPpidCr-lV0b-3fChlyoMRluQhEMlSMN5S1UJiDaEKIFRpzBPgZQ6eb0_njY2O2g1uCEoOzOd_3FmK3v_UzJKSFVNwbw5GgT_Y4Q4yJ2JGqxVDlJ6p7hFhUuWirbIXv2D3n-7I9WrdAHjOp_O1ZOpvChTwRGnJU7U8h4qPS3sjE691Jm0PxO8nQkSM8CvoVdjjHJ9vfl_9ur7nH19wm5B2WEbvR2nJoxzsDyAOvgYA3R3ScZITqNwmw05jYI8jkKSvTgt0J3otvfpH-4bAxQ</recordid><startdate>20170223</startdate><enddate>20170223</enddate><creator>Ipsaro, Jonathan J</creator><creator>Shen, Chen</creator><creator>Arai, Eri</creator><creator>Xu, Yali</creator><creator>Kinney, Justin B</creator><creator>Joshua-Tor, Leemor</creator><creator>Vakoc, Christopher R</creator><creator>Shi, Junwei</creator><general>Public Library of Science</general><general>Public Library of Science (PLoS)</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>IOV</scope><scope>ISR</scope><scope>3V.</scope><scope>7QG</scope><scope>7QL</scope><scope>7QO</scope><scope>7RV</scope><scope>7SN</scope><scope>7SS</scope><scope>7T5</scope><scope>7TG</scope><scope>7TM</scope><scope>7U9</scope><scope>7X2</scope><scope>7X7</scope><scope>7XB</scope><scope>88E</scope><scope>8AO</scope><scope>8C1</scope><scope>8FD</scope><scope>8FE</scope><scope>8FG</scope><scope>8FH</scope><scope>8FI</scope><scope>8FJ</scope><scope>8FK</scope><scope>ABJCF</scope><scope>ABUWG</scope><scope>AEUYN</scope><scope>AFKRA</scope><scope>ARAPS</scope><scope>ATCPS</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BGLVJ</scope><scope>BHPHI</scope><scope>C1K</scope><scope>CCPQU</scope><scope>D1I</scope><scope>DWQXO</scope><scope>FR3</scope><scope>FYUFA</scope><scope>GHDGH</scope><scope>GNUQQ</scope><scope>H94</scope><scope>HCIFZ</scope><scope>K9.</scope><scope>KB.</scope><scope>KB0</scope><scope>KL.</scope><scope>L6V</scope><scope>LK8</scope><scope>M0K</scope><scope>M0S</scope><scope>M1P</scope><scope>M7N</scope><scope>M7P</scope><scope>M7S</scope><scope>NAPCQ</scope><scope>P5Z</scope><scope>P62</scope><scope>P64</scope><scope>PATMY</scope><scope>PDBOC</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PTHSS</scope><scope>PYCSY</scope><scope>RC3</scope><scope>5PM</scope><scope>DOA</scope><orcidid>https://orcid.org/0000-0002-8427-6316</orcidid></search><sort><creationdate>20170223</creationdate><title>Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis</title><author>Ipsaro, Jonathan J ; Shen, Chen ; Arai, Eri ; Xu, Yali ; Kinney, Justin B ; Joshua-Tor, Leemor ; Vakoc, Christopher R ; Shi, Junwei</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c725t-3463d2c85135e727f723501b40e9140ec530936affae559fcb9dbca4ce6dbcf63</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Alleles</topic><topic>Animals</topic><topic>Antineoplastic Agents - pharmacology</topic><topic>Benzimidazoles - pharmacology</topic><topic>Biology</topic><topic>Biology and Life Sciences</topic><topic>Cancer</topic><topic>Cell Line</topic><topic>Cell Line, Tumor</topic><topic>Cell Proliferation - drug effects</topic><topic>Clonal deletion</topic><topic>Cloning</topic><topic>Clustered Regularly Interspaced Short Palindromic Repeats</topic><topic>CRISPR</topic><topic>CRISPR-Cas Systems</topic><topic>Deoxyribonucleic acid</topic><topic>Developmental stages</topic><topic>Discovery tools</topic><topic>DNA</topic><topic>DNA methylation</topic><topic>Drug development</topic><topic>Drug resistance</topic><topic>Drug Resistance, Neoplasm</topic><topic>Engineering and Technology</topic><topic>Enhancer of Zeste Homolog 2 Protein - genetics</topic><topic>Enhancer of Zeste Homolog 2 Protein - metabolism</topic><topic>Genes</topic><topic>Genetic aspects</topic><topic>Health aspects</topic><topic>HEK293 Cells</topic><topic>Humans</topic><topic>INDEL Mutation</topic><topic>Inhibitors</topic><topic>Insertion</topic><topic>Laboratories</topic><topic>Leukemia</topic><topic>Loci</topic><topic>Lysine</topic><topic>Medical research</topic><topic>Methyltransferases - 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Unfortunately, for many therapeutic targets such alleles are not available. To address this issue, we evaluated whether CRISPR-Cas9-mediated insertion/deletion (indel) mutagenesis can produce drug-resistance alleles at endogenous loci. This method takes advantage of the heterogeneous in-frame alleles produced following Cas9-mediated DNA cleavage, which we show can generate rare alleles that confer resistance to the growth-arrest caused by chemical inhibitors. We used this approach to identify novel resistance alleles of two lysine methyltransferases, DOT1L and EZH2, which are each essential for the growth of MLL-fusion leukemia cells. We biochemically characterized the DOT1L mutation, showing that it is significantly more active than the wild-type enzyme. These findings validate the on-target anti-leukemia activities of existing DOT1L and EZH2 inhibitors and reveal a simple method for deriving drug-resistance alleles for novel targets, which may have utility during early stages of drug development.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>28231254</pmid><doi>10.1371/journal.pone.0172177</doi><tpages>e0172177</tpages><orcidid>https://orcid.org/0000-0002-8427-6316</orcidid><oa>free_for_read</oa></addata></record> |
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| subjects | Alleles Animals Antineoplastic Agents - pharmacology Benzimidazoles - pharmacology Biology Biology and Life Sciences Cancer Cell Line Cell Line, Tumor Cell Proliferation - drug effects Clonal deletion Cloning Clustered Regularly Interspaced Short Palindromic Repeats CRISPR CRISPR-Cas Systems Deoxyribonucleic acid Developmental stages Discovery tools DNA DNA methylation Drug development Drug resistance Drug Resistance, Neoplasm Engineering and Technology Enhancer of Zeste Homolog 2 Protein - genetics Enhancer of Zeste Homolog 2 Protein - metabolism Genes Genetic aspects Health aspects HEK293 Cells Humans INDEL Mutation Inhibitors Insertion Laboratories Leukemia Loci Lysine Medical research Methyltransferases - genetics Methyltransferases - metabolism Mice Models, Molecular Mutagenesis Mutation Neoplasms - drug therapy Neoplasms - genetics Neoplasms - metabolism Plasmids Proteins Research and Analysis Methods |
| title | Rapid generation of drug-resistance alleles at endogenous loci using CRISPR-Cas9 indel mutagenesis |
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