Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1
The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM),...
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| Veröffentlicht in: | Molecular cell 2017-07, Vol.67 (1), p.139-147.e2 |
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| creator | Nishimasu, Hiroshi Yamano, Takashi Gao, Linyi Zhang, Feng Ishitani, Ryuichiro Nureki, Osamu |
| description | The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
[Display omitted]
•Crystal structures of the AsCpf1 RVR and RR variants at 2.0 Å resolution•Structural basis for the TATV PAM recognition by the RVR variant•Structural basis for the TYCV PAM recognition by the RR variant
Variants of AsCpf1 with altered PAM specificities have been derived. Nishimasu et al. determined the high-resolution crystal structures of two of these variants bound to a crRNA and its target DNA, thereby explaining their altered PAM specificities. |
| doi_str_mv | 10.1016/j.molcel.2017.04.019 |
| format | Article |
| fullrecord | <record><control><sourceid>proquest_pubme</sourceid><recordid>TN_cdi_pubmedcentral_primary_oai_pubmedcentral_nih_gov_5957533</recordid><sourceformat>XML</sourceformat><sourcesystem>PC</sourcesystem><els_id>S1097276517302757</els_id><sourcerecordid>2000438381</sourcerecordid><originalsourceid>FETCH-LOGICAL-c562t-46c5893cb1bd7153526e32cdf5c066890af1dbfcb3d273f588e3d0abd084a933</originalsourceid><addsrcrecordid>eNqFUU1v1DAQtRCIlsI_QChHLgn-TnxBWlaFFhVRbXu3HHuy9SobL7ZTqf8eb3cpcIHTjDRv3rw3D6G3BDcEE_lh02zDaGFsKCZtg3mDiXqGTglWbc2J5M-PPW2lOEGvUtpgTLjo1Et0QjuhSidP0debHGeb52jG6pNJPlVDiFW-g2oxZojgquvFt2oFNqwnn32Yqv6hOp_WfoLH6XJ1eXO9qpe7gbxGLwYzJnhzrGfo9vP57fKivvr-5XK5uKqtkDTXXNpymtme9K4lggkqgVHrBmGxlJ3CZiCuH2zPHG3ZILoOmMOmd7jjRjF2hj4eaHdzvwVnYcpFvN5FvzXxQQfj9d-Tyd_pdbjXxXIr2J7g_ZEghh8zpKy3PpVHjmaCMCdNMcacdawj_4USVUQxypUqUH6A2hhSijA8KSJY7wPTG30ITO8D05jrElhZe_enm6elXwn9tgvlpfceok7Ww2TB-Qg2axf8vy_8BBedqHw</addsrcrecordid><sourcetype>Open Access Repository</sourcetype><iscdi>true</iscdi><recordtype>article</recordtype><pqid>1908432499</pqid></control><display><type>article</type><title>Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1</title><source>MEDLINE</source><source>Cell Press Free Archives</source><source>Elsevier ScienceDirect Journals</source><source>EZB-FREE-00999 freely available EZB journals</source><source>Free Full-Text Journals in Chemistry</source><creator>Nishimasu, Hiroshi ; Yamano, Takashi ; Gao, Linyi ; Zhang, Feng ; Ishitani, Ryuichiro ; Nureki, Osamu</creator><creatorcontrib>Nishimasu, Hiroshi ; Yamano, Takashi ; Gao, Linyi ; Zhang, Feng ; Ishitani, Ryuichiro ; Nureki, Osamu</creatorcontrib><description>The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
[Display omitted]
•Crystal structures of the AsCpf1 RVR and RR variants at 2.0 Å resolution•Structural basis for the TATV PAM recognition by the RVR variant•Structural basis for the TYCV PAM recognition by the RR variant
Variants of AsCpf1 with altered PAM specificities have been derived. Nishimasu et al. determined the high-resolution crystal structures of two of these variants bound to a crRNA and its target DNA, thereby explaining their altered PAM specificities.</description><identifier>ISSN: 1097-2765</identifier><identifier>EISSN: 1097-4164</identifier><identifier>DOI: 10.1016/j.molcel.2017.04.019</identifier><identifier>PMID: 28595896</identifier><language>eng</language><publisher>United States: Elsevier Inc</publisher><subject>Acidaminococcus ; Acidaminococcus - enzymology ; Acidaminococcus - genetics ; Bacterial Proteins - chemistry ; Bacterial Proteins - genetics ; Bacterial Proteins - metabolism ; Cas12a ; Clustered Regularly Interspaced Short Palindromic Repeats ; Cpf1 ; CRISPR-Associated Proteins - chemistry ; CRISPR-Associated Proteins - genetics ; CRISPR-Associated Proteins - metabolism ; CRISPR-Cas system ; CRISPR-Cas Systems ; crystal structure ; DNA ; DNA, Bacterial - chemistry ; DNA, Bacterial - genetics ; DNA, Bacterial - metabolism ; engineering ; Escherichia coli - enzymology ; Escherichia coli - genetics ; Gene Editing ; Models, Molecular ; Mutation ; Nucleic Acid Conformation ; Nucleic Acid Heteroduplexes ; nucleotide sequences ; nucleotides ; Protein Binding ; Protein Conformation ; protospacer adjacent motif ; RNA ; RNA - chemistry ; RNA - genetics ; RNA - metabolism ; Structure-Activity Relationship</subject><ispartof>Molecular cell, 2017-07, Vol.67 (1), p.139-147.e2</ispartof><rights>2017 Elsevier Inc.</rights><rights>Copyright © 2017 Elsevier Inc. All rights reserved.</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c562t-46c5893cb1bd7153526e32cdf5c066890af1dbfcb3d273f588e3d0abd084a933</citedby><cites>FETCH-LOGICAL-c562t-46c5893cb1bd7153526e32cdf5c066890af1dbfcb3d273f588e3d0abd084a933</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://www.sciencedirect.com/science/article/pii/S1097276517302757$$EHTML$$P50$$Gelsevier$$Hfree_for_read</linktohtml><link.rule.ids>230,314,776,780,881,3537,27901,27902,65306</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/28595896$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Nishimasu, Hiroshi</creatorcontrib><creatorcontrib>Yamano, Takashi</creatorcontrib><creatorcontrib>Gao, Linyi</creatorcontrib><creatorcontrib>Zhang, Feng</creatorcontrib><creatorcontrib>Ishitani, Ryuichiro</creatorcontrib><creatorcontrib>Nureki, Osamu</creatorcontrib><title>Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1</title><title>Molecular cell</title><addtitle>Mol Cell</addtitle><description>The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
[Display omitted]
•Crystal structures of the AsCpf1 RVR and RR variants at 2.0 Å resolution•Structural basis for the TATV PAM recognition by the RVR variant•Structural basis for the TYCV PAM recognition by the RR variant
Variants of AsCpf1 with altered PAM specificities have been derived. Nishimasu et al. determined the high-resolution crystal structures of two of these variants bound to a crRNA and its target DNA, thereby explaining their altered PAM specificities.</description><subject>Acidaminococcus</subject><subject>Acidaminococcus - enzymology</subject><subject>Acidaminococcus - genetics</subject><subject>Bacterial Proteins - chemistry</subject><subject>Bacterial Proteins - genetics</subject><subject>Bacterial Proteins - metabolism</subject><subject>Cas12a</subject><subject>Clustered Regularly Interspaced Short Palindromic Repeats</subject><subject>Cpf1</subject><subject>CRISPR-Associated Proteins - chemistry</subject><subject>CRISPR-Associated Proteins - genetics</subject><subject>CRISPR-Associated Proteins - metabolism</subject><subject>CRISPR-Cas system</subject><subject>CRISPR-Cas Systems</subject><subject>crystal structure</subject><subject>DNA</subject><subject>DNA, Bacterial - chemistry</subject><subject>DNA, Bacterial - genetics</subject><subject>DNA, Bacterial - metabolism</subject><subject>engineering</subject><subject>Escherichia coli - enzymology</subject><subject>Escherichia coli - genetics</subject><subject>Gene Editing</subject><subject>Models, Molecular</subject><subject>Mutation</subject><subject>Nucleic Acid Conformation</subject><subject>Nucleic Acid Heteroduplexes</subject><subject>nucleotide sequences</subject><subject>nucleotides</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>protospacer adjacent motif</subject><subject>RNA</subject><subject>RNA - chemistry</subject><subject>RNA - genetics</subject><subject>RNA - metabolism</subject><subject>Structure-Activity Relationship</subject><issn>1097-2765</issn><issn>1097-4164</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2017</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFUU1v1DAQtRCIlsI_QChHLgn-TnxBWlaFFhVRbXu3HHuy9SobL7ZTqf8eb3cpcIHTjDRv3rw3D6G3BDcEE_lh02zDaGFsKCZtg3mDiXqGTglWbc2J5M-PPW2lOEGvUtpgTLjo1Et0QjuhSidP0debHGeb52jG6pNJPlVDiFW-g2oxZojgquvFt2oFNqwnn32Yqv6hOp_WfoLH6XJ1eXO9qpe7gbxGLwYzJnhzrGfo9vP57fKivvr-5XK5uKqtkDTXXNpymtme9K4lggkqgVHrBmGxlJ3CZiCuH2zPHG3ZILoOmMOmd7jjRjF2hj4eaHdzvwVnYcpFvN5FvzXxQQfj9d-Tyd_pdbjXxXIr2J7g_ZEghh8zpKy3PpVHjmaCMCdNMcacdawj_4USVUQxypUqUH6A2hhSijA8KSJY7wPTG30ITO8D05jrElhZe_enm6elXwn9tgvlpfceok7Ww2TB-Qg2axf8vy_8BBedqHw</recordid><startdate>20170706</startdate><enddate>20170706</enddate><creator>Nishimasu, Hiroshi</creator><creator>Yamano, Takashi</creator><creator>Gao, Linyi</creator><creator>Zhang, Feng</creator><creator>Ishitani, Ryuichiro</creator><creator>Nureki, Osamu</creator><general>Elsevier Inc</general><scope>6I.</scope><scope>AAFTH</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>7X8</scope><scope>7S9</scope><scope>L.6</scope><scope>5PM</scope></search><sort><creationdate>20170706</creationdate><title>Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1</title><author>Nishimasu, Hiroshi ; Yamano, Takashi ; Gao, Linyi ; Zhang, Feng ; Ishitani, Ryuichiro ; Nureki, Osamu</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c562t-46c5893cb1bd7153526e32cdf5c066890af1dbfcb3d273f588e3d0abd084a933</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2017</creationdate><topic>Acidaminococcus</topic><topic>Acidaminococcus - enzymology</topic><topic>Acidaminococcus - genetics</topic><topic>Bacterial Proteins - chemistry</topic><topic>Bacterial Proteins - genetics</topic><topic>Bacterial Proteins - metabolism</topic><topic>Cas12a</topic><topic>Clustered Regularly Interspaced Short Palindromic Repeats</topic><topic>Cpf1</topic><topic>CRISPR-Associated Proteins - chemistry</topic><topic>CRISPR-Associated Proteins - genetics</topic><topic>CRISPR-Associated Proteins - metabolism</topic><topic>CRISPR-Cas system</topic><topic>CRISPR-Cas Systems</topic><topic>crystal structure</topic><topic>DNA</topic><topic>DNA, Bacterial - chemistry</topic><topic>DNA, Bacterial - genetics</topic><topic>DNA, Bacterial - metabolism</topic><topic>engineering</topic><topic>Escherichia coli - enzymology</topic><topic>Escherichia coli - genetics</topic><topic>Gene Editing</topic><topic>Models, Molecular</topic><topic>Mutation</topic><topic>Nucleic Acid Conformation</topic><topic>Nucleic Acid Heteroduplexes</topic><topic>nucleotide sequences</topic><topic>nucleotides</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>protospacer adjacent motif</topic><topic>RNA</topic><topic>RNA - chemistry</topic><topic>RNA - genetics</topic><topic>RNA - metabolism</topic><topic>Structure-Activity Relationship</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Nishimasu, Hiroshi</creatorcontrib><creatorcontrib>Yamano, Takashi</creatorcontrib><creatorcontrib>Gao, Linyi</creatorcontrib><creatorcontrib>Zhang, Feng</creatorcontrib><creatorcontrib>Ishitani, Ryuichiro</creatorcontrib><creatorcontrib>Nureki, Osamu</creatorcontrib><collection>ScienceDirect Open Access Titles</collection><collection>Elsevier:ScienceDirect:Open Access</collection><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><collection>AGRICOLA</collection><collection>AGRICOLA - Academic</collection><collection>PubMed Central (Full Participant titles)</collection><jtitle>Molecular cell</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Nishimasu, Hiroshi</au><au>Yamano, Takashi</au><au>Gao, Linyi</au><au>Zhang, Feng</au><au>Ishitani, Ryuichiro</au><au>Nureki, Osamu</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1</atitle><jtitle>Molecular cell</jtitle><addtitle>Mol Cell</addtitle><date>2017-07-06</date><risdate>2017</risdate><volume>67</volume><issue>1</issue><spage>139</spage><epage>147.e2</epage><pages>139-147.e2</pages><issn>1097-2765</issn><eissn>1097-4164</eissn><abstract>The RNA-guided Cpf1 nuclease cleaves double-stranded DNA targets complementary to the CRISPR RNA (crRNA), and it has been harnessed for genome editing technologies. Recently, Acidaminococcus sp. BV3L6 (AsCpf1) was engineered to recognize altered DNA sequences as the protospacer adjacent motif (PAM), thereby expanding the target range of Cpf1-mediated genome editing. Whereas wild-type AsCpf1 recognizes the TTTV PAM, the RVR (S542R/K548V/N552R) and RR (S542R/K607R) variants can efficiently recognize the TATV and TYCV PAMs, respectively. However, their PAM recognition mechanisms remained unknown. Here we present the 2.0 Å resolution crystal structures of the RVR and RR variants bound to a crRNA and its target DNA. The structures revealed that the RVR and RR variants primarily recognize the PAM-complementary nucleotides via the substituted residues. Our high-resolution structures delineated the altered PAM recognition mechanisms of the AsCpf1 variants, providing a basis for the further engineering of CRISPR-Cpf1.
[Display omitted]
•Crystal structures of the AsCpf1 RVR and RR variants at 2.0 Å resolution•Structural basis for the TATV PAM recognition by the RVR variant•Structural basis for the TYCV PAM recognition by the RR variant
Variants of AsCpf1 with altered PAM specificities have been derived. Nishimasu et al. determined the high-resolution crystal structures of two of these variants bound to a crRNA and its target DNA, thereby explaining their altered PAM specificities.</abstract><cop>United States</cop><pub>Elsevier Inc</pub><pmid>28595896</pmid><doi>10.1016/j.molcel.2017.04.019</doi><tpages>9</tpages><oa>free_for_read</oa></addata></record> |
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| subjects | Acidaminococcus Acidaminococcus - enzymology Acidaminococcus - genetics Bacterial Proteins - chemistry Bacterial Proteins - genetics Bacterial Proteins - metabolism Cas12a Clustered Regularly Interspaced Short Palindromic Repeats Cpf1 CRISPR-Associated Proteins - chemistry CRISPR-Associated Proteins - genetics CRISPR-Associated Proteins - metabolism CRISPR-Cas system CRISPR-Cas Systems crystal structure DNA DNA, Bacterial - chemistry DNA, Bacterial - genetics DNA, Bacterial - metabolism engineering Escherichia coli - enzymology Escherichia coli - genetics Gene Editing Models, Molecular Mutation Nucleic Acid Conformation Nucleic Acid Heteroduplexes nucleotide sequences nucleotides Protein Binding Protein Conformation protospacer adjacent motif RNA RNA - chemistry RNA - genetics RNA - metabolism Structure-Activity Relationship |
| title | Structural Basis for the Altered PAM Recognition by Engineered CRISPR-Cpf1 |
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