The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a
Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as obser...
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| Veröffentlicht in: | Genes 2019-02, Vol.10 (2), p.169 |
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| creator | van Aelst, Kara Martínez-Santiago, Carlos J Cross, Stephen J Szczelkun, Mark D |
| description | Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by
bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with
DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies. |
| doi_str_mv | 10.3390/genes10020169 |
| format | Article |
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DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.</description><identifier>ISSN: 2073-4425</identifier><identifier>EISSN: 2073-4425</identifier><identifier>DOI: 10.3390/genes10020169</identifier><identifier>PMID: 30813348</identifier><language>eng</language><publisher>Switzerland: MDPI AG</publisher><subject>Bacterial Proteins - metabolism ; Circular DNA ; CRISPR ; CRISPR-Cas Systems ; DNA, Superhelical - chemistry ; DNA, Superhelical - metabolism ; Endodeoxyribonucleases - metabolism ; Enzymes ; Kinetics ; Mutation ; R-Loop Structures ; Supercoiling</subject><ispartof>Genes, 2019-02, Vol.10 (2), p.169</ispartof><rights>2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>2019 by the authors. 2019</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c415t-64e2c771112cd84b4f1e97040e489c6ddd5f4bf9517f93b212a49307e8cebbcf3</citedby><cites>FETCH-LOGICAL-c415t-64e2c771112cd84b4f1e97040e489c6ddd5f4bf9517f93b212a49307e8cebbcf3</cites><orcidid>0000-0003-3565-0479 ; 0000-0002-2501-6602</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/PMC6409811/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC6409811/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,881,27901,27902,53766,53768</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/30813348$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>van Aelst, Kara</creatorcontrib><creatorcontrib>Martínez-Santiago, Carlos J</creatorcontrib><creatorcontrib>Cross, Stephen J</creatorcontrib><creatorcontrib>Szczelkun, Mark D</creatorcontrib><title>The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a</title><title>Genes</title><addtitle>Genes (Basel)</addtitle><description>Here we explored the mechanism of R-loop formation and DNA cleavage by type V CRISPR Cas12a (formerly known as Cpf1). We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by
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DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.</description><subject>Bacterial Proteins - metabolism</subject><subject>Circular DNA</subject><subject>CRISPR</subject><subject>CRISPR-Cas Systems</subject><subject>DNA, Superhelical - chemistry</subject><subject>DNA, Superhelical - metabolism</subject><subject>Endodeoxyribonucleases - metabolism</subject><subject>Enzymes</subject><subject>Kinetics</subject><subject>Mutation</subject><subject>R-Loop Structures</subject><subject>Supercoiling</subject><issn>2073-4425</issn><issn>2073-4425</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2019</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>BENPR</sourceid><recordid>eNpdkc1LAzEQxYMoKtWjVwl48bKaSbLd3Ysga_2AolLrOWSzk7qy3dRkW-h_b-oX6lxmYH7zeMMj5AjYmRAFO59hhwEY4wyGxRbZ5ywTiZQ83f4175HDEF5ZLBlBlu6SPcFyEELm-6SZviAdWYump87Sq_tLOnUL17rZmrqOPlQB_QprOtE9hg0xScbOLei183PdNxHRXf1x9tT7zVi2qFd6hrRa03Jy9_Q4oaUOwPUB2bG6DXj41Qfk-Xo0LW-T8cPNXXk5ToyEtE-GErnJMgDgps5lJS1gkUXrKPPCDOu6Tq2sbJFCZgtR8agsC8EyzA1WlbFiQC4-dRfLao61wS4aa9XCN3Pt18rpRv3ddM2LmrmVGkpW5ABR4PRLwLu3JYZezZtgsG11h24ZFIc8Y1wCkxE9-Ye-uqXv4nuKpzJPIeXR3IAkn5TxLgSP9scMMLXJUf3JMfLHvz_4ob9TE-8PR5bf</recordid><startdate>20190222</startdate><enddate>20190222</enddate><creator>van Aelst, Kara</creator><creator>Martínez-Santiago, Carlos J</creator><creator>Cross, Stephen J</creator><creator>Szczelkun, Mark D</creator><general>MDPI AG</general><general>MDPI</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>8FD</scope><scope>8FE</scope><scope>8FH</scope><scope>ABUWG</scope><scope>AFKRA</scope><scope>AZQEC</scope><scope>BBNVY</scope><scope>BENPR</scope><scope>BHPHI</scope><scope>CCPQU</scope><scope>DWQXO</scope><scope>FR3</scope><scope>GNUQQ</scope><scope>HCIFZ</scope><scope>LK8</scope><scope>M7P</scope><scope>P64</scope><scope>PIMPY</scope><scope>PQEST</scope><scope>PQQKQ</scope><scope>PQUKI</scope><scope>PRINS</scope><scope>RC3</scope><scope>7X8</scope><scope>5PM</scope><orcidid>https://orcid.org/0000-0003-3565-0479</orcidid><orcidid>https://orcid.org/0000-0002-2501-6602</orcidid></search><sort><creationdate>20190222</creationdate><title>The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a</title><author>van Aelst, Kara ; 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We first used a single-molecule magnetic tweezers (MT) assay to show that R-loop formation by
bacterium ND2006 Cas12a is significantly enhanced by negative DNA supercoiling, as observed previously with
DGCC7710 CRISPR3 Cas9. Consistent with the MT data, the apparent rate of cleavage of supercoiled plasmid DNA was observed to be >50-fold faster than the apparent rates for linear DNA or nicked circular DNA because of topology-dependent differences in R-loop formation kinetics. Taking the differences into account, the cleavage data for all substrates can be fitted with the same apparent rate constants for the two strand-cleavage steps, with the first event >15-fold faster than the second. By independently following the ensemble cleavage of the non-target strand (NTS) and target strand (TS), we could show that the faster rate is due to NTS cleavage, the slower rate due to TS cleavage, as expected from previous studies.</abstract><cop>Switzerland</cop><pub>MDPI AG</pub><pmid>30813348</pmid><doi>10.3390/genes10020169</doi><orcidid>https://orcid.org/0000-0003-3565-0479</orcidid><orcidid>https://orcid.org/0000-0002-2501-6602</orcidid><oa>free_for_read</oa></addata></record> |
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| source | Electronic Journals Library; MDPI - Multidisciplinary Digital Publishing Institute; MEDLINE; PubMed Central; PubMed Central Open Access |
| subjects | Bacterial Proteins - metabolism Circular DNA CRISPR CRISPR-Cas Systems DNA, Superhelical - chemistry DNA, Superhelical - metabolism Endodeoxyribonucleases - metabolism Enzymes Kinetics Mutation R-Loop Structures Supercoiling |
| title | The Effect of DNA Topology on Observed Rates of R-Loop Formation and DNA Strand Cleavage by CRISPR Cas12a |
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