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combinatorial approach to create artificial homing endonucleases cleaving chosen sequences
Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of...
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Published in: | Nucleic acids research 2006-12, Vol.34 (22), p.e149-e149 |
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creator | Smith, Julianne Grizot, Sylvestre Arnould, Sylvain Duclert, Aymeric Epinat, Jean-Charles Chames, Patrick Prieto, Jesús Redondo, Pilar Blanco, Francisco J Bravo, Jerónimo Montoya, Guillermo Pâques, Frédéric Duchateau, Philippe |
description | Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities. |
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These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.</description><identifier>ISSN: 0305-1048</identifier><identifier>EISSN: 1362-4962</identifier><identifier>DOI: 10.1093/nar/gkl720</identifier><identifier>PMID: 17130168</identifier><identifier>CODEN: NARHAD</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>Chlamydomonas ; Data Interpretation, Statistical ; Dimerization ; DNA - metabolism ; DNA Restriction Enzymes - chemistry ; DNA Restriction Enzymes - genetics ; DNA Restriction Enzymes - metabolism ; Genes, RAG-1 ; Humans ; Methods Online ; Mutation ; Nucleotides - metabolism ; Peptide Library ; Protein Engineering - methods ; Protein Structure, Tertiary ; Substrate Specificity</subject><ispartof>Nucleic acids research, 2006-12, Vol.34 (22), p.e149-e149</ispartof><rights>2006 The Author(s). 2006</rights><rights>Copyright Oxford University Press(England) Dec 2006</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c554t-c824548f5e64a670a0ad19af45c1abd758344f02a2b8fa75fc27b7b05df6ec473</citedby><cites>FETCH-LOGICAL-c554t-c824548f5e64a670a0ad19af45c1abd758344f02a2b8fa75fc27b7b05df6ec473</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/PMC1702487/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1702487/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,727,780,784,885,1604,27924,27925,53791,53793</link.rule.ids><linktorsrc>$$Uhttps://dx.doi.org/10.1093/nar/gkl720$$EView_record_in_Oxford_University_Press$$FView_record_in_$$GOxford_University_Press</linktorsrc><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17130168$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Smith, Julianne</creatorcontrib><creatorcontrib>Grizot, Sylvestre</creatorcontrib><creatorcontrib>Arnould, Sylvain</creatorcontrib><creatorcontrib>Duclert, Aymeric</creatorcontrib><creatorcontrib>Epinat, Jean-Charles</creatorcontrib><creatorcontrib>Chames, Patrick</creatorcontrib><creatorcontrib>Prieto, Jesús</creatorcontrib><creatorcontrib>Redondo, Pilar</creatorcontrib><creatorcontrib>Blanco, Francisco J</creatorcontrib><creatorcontrib>Bravo, Jerónimo</creatorcontrib><creatorcontrib>Montoya, Guillermo</creatorcontrib><creatorcontrib>Pâques, Frédéric</creatorcontrib><creatorcontrib>Duchateau, Philippe</creatorcontrib><title>combinatorial approach to create artificial homing endonucleases cleaving chosen sequences</title><title>Nucleic acids research</title><addtitle>Nucleic Acids Res</addtitle><description>Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. 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These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.</description><subject>Chlamydomonas</subject><subject>Data Interpretation, Statistical</subject><subject>Dimerization</subject><subject>DNA - metabolism</subject><subject>DNA Restriction Enzymes - chemistry</subject><subject>DNA Restriction Enzymes - genetics</subject><subject>DNA Restriction Enzymes - metabolism</subject><subject>Genes, RAG-1</subject><subject>Humans</subject><subject>Methods Online</subject><subject>Mutation</subject><subject>Nucleotides - metabolism</subject><subject>Peptide Library</subject><subject>Protein Engineering - methods</subject><subject>Protein Structure, Tertiary</subject><subject>Substrate Specificity</subject><issn>0305-1048</issn><issn>1362-4962</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2006</creationdate><recordtype>article</recordtype><recordid>eNqFkU2L1TAUhoMozp3RjT9AizAuhDonab66EWTwCwZc6GzchNM0ubdjm3SSdsB_b0svfi10dQ7nPDyc5CXkCYVXFOrqImC62H_rFYN7ZEcryUpeS3af7KACUVLg-oSc5nwDQDkV_CE5oYpWQKXeka82Dk0XcIqpw77AcUwR7aGYYmGTw8kVmKbOd3bdHuLQhX3hQhvDbHuH2eVirXfr2B5idqHI7nZ2wbr8iDzw2Gf3-FjPyPW7t18uP5RXn95_vHxzVVoh-FRazbjg2gsnOUoFCNjSGj0XlmLTKqErzj0wZI32qIS3TDWqAdF66SxX1Rl5vXnHuRlca12YEvZmTN2A6buJ2Jk_N6E7mH28M1QB43oVvDgKUlxuz5MZumxd32Nwcc5GaiYF0_q_IK0FZZrKBXz-F3gT5xSWXzAMQFageL1ALzfIpphzcv7nyRTMGqxZgjVbsAv89PdH_kKPSS7A-QbEefy36NnGeYwG96nL5vozg9VCGadL8wNc7rdc</recordid><startdate>20061201</startdate><enddate>20061201</enddate><creator>Smith, Julianne</creator><creator>Grizot, Sylvestre</creator><creator>Arnould, Sylvain</creator><creator>Duclert, Aymeric</creator><creator>Epinat, Jean-Charles</creator><creator>Chames, Patrick</creator><creator>Prieto, Jesús</creator><creator>Redondo, Pilar</creator><creator>Blanco, Francisco J</creator><creator>Bravo, Jerónimo</creator><creator>Montoya, Guillermo</creator><creator>Pâques, Frédéric</creator><creator>Duchateau, Philippe</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>FBQ</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>7QL</scope><scope>7QO</scope><scope>7QP</scope><scope>7QR</scope><scope>7SS</scope><scope>7TK</scope><scope>7TM</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope></search><sort><creationdate>20061201</creationdate><title>combinatorial approach to create artificial homing endonucleases cleaving chosen sequences</title><author>Smith, Julianne ; Grizot, Sylvestre ; Arnould, Sylvain ; Duclert, Aymeric ; Epinat, Jean-Charles ; Chames, Patrick ; Prieto, Jesús ; Redondo, Pilar ; Blanco, Francisco J ; Bravo, Jerónimo ; Montoya, Guillermo ; Pâques, Frédéric ; Duchateau, Philippe</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c554t-c824548f5e64a670a0ad19af45c1abd758344f02a2b8fa75fc27b7b05df6ec473</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2006</creationdate><topic>Chlamydomonas</topic><topic>Data Interpretation, Statistical</topic><topic>Dimerization</topic><topic>DNA - metabolism</topic><topic>DNA Restriction Enzymes - chemistry</topic><topic>DNA Restriction Enzymes - genetics</topic><topic>DNA Restriction Enzymes - metabolism</topic><topic>Genes, RAG-1</topic><topic>Humans</topic><topic>Methods Online</topic><topic>Mutation</topic><topic>Nucleotides - metabolism</topic><topic>Peptide Library</topic><topic>Protein Engineering - methods</topic><topic>Protein Structure, Tertiary</topic><topic>Substrate Specificity</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Smith, Julianne</creatorcontrib><creatorcontrib>Grizot, Sylvestre</creatorcontrib><creatorcontrib>Arnould, Sylvain</creatorcontrib><creatorcontrib>Duclert, Aymeric</creatorcontrib><creatorcontrib>Epinat, Jean-Charles</creatorcontrib><creatorcontrib>Chames, Patrick</creatorcontrib><creatorcontrib>Prieto, Jesús</creatorcontrib><creatorcontrib>Redondo, Pilar</creatorcontrib><creatorcontrib>Blanco, Francisco J</creatorcontrib><creatorcontrib>Bravo, Jerónimo</creatorcontrib><creatorcontrib>Montoya, Guillermo</creatorcontrib><creatorcontrib>Pâques, Frédéric</creatorcontrib><creatorcontrib>Duchateau, Philippe</creatorcontrib><collection>AGRIS</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Bacteriology Abstracts (Microbiology B)</collection><collection>Biotechnology Research Abstracts</collection><collection>Calcium & Calcified Tissue Abstracts</collection><collection>Chemoreception Abstracts</collection><collection>Entomology Abstracts (Full archive)</collection><collection>Neurosciences Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Virology and AIDS Abstracts</collection><collection>Technology Research Database</collection><collection>Environmental Sciences and Pollution Management</collection><collection>Engineering Research Database</collection><collection>AIDS and Cancer Research Abstracts</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Algology Mycology and Protozoology Abstracts (Microbiology C)</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>Genetics Abstracts</collection><collection>MEDLINE - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Nucleic acids research</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext_linktorsrc</fulltext></delivery><addata><au>Smith, Julianne</au><au>Grizot, Sylvestre</au><au>Arnould, Sylvain</au><au>Duclert, Aymeric</au><au>Epinat, Jean-Charles</au><au>Chames, Patrick</au><au>Prieto, Jesús</au><au>Redondo, Pilar</au><au>Blanco, Francisco J</au><au>Bravo, Jerónimo</au><au>Montoya, Guillermo</au><au>Pâques, Frédéric</au><au>Duchateau, Philippe</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>combinatorial approach to create artificial homing endonucleases cleaving chosen sequences</atitle><jtitle>Nucleic acids research</jtitle><addtitle>Nucleic Acids Res</addtitle><date>2006-12-01</date><risdate>2006</risdate><volume>34</volume><issue>22</issue><spage>e149</spage><epage>e149</epage><pages>e149-e149</pages><issn>0305-1048</issn><eissn>1362-4962</eissn><coden>NARHAD</coden><abstract>Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>17130168</pmid><doi>10.1093/nar/gkl720</doi><oa>free_for_read</oa></addata></record> |
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subjects | Chlamydomonas Data Interpretation, Statistical Dimerization DNA - metabolism DNA Restriction Enzymes - chemistry DNA Restriction Enzymes - genetics DNA Restriction Enzymes - metabolism Genes, RAG-1 Humans Methods Online Mutation Nucleotides - metabolism Peptide Library Protein Engineering - methods Protein Structure, Tertiary Substrate Specificity |
title | combinatorial approach to create artificial homing endonucleases cleaving chosen sequences |
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