DNA stretching induces Cas9 off-target activity.
Identifieur interne : 000928 ( PubMed/Corpus ); précédent : 000927; suivant : 000929DNA stretching induces Cas9 off-target activity.
Auteurs : Matthew D. Newton ; Benjamin J. Taylor ; Rosalie P C. Driessen ; Leonie Roos ; Nevena Cvetesic ; Shenaz Allyjaun ; Boris Lenhard ; Maria Emanuela Cuomo ; David S. RuedaSource :
- Nature structural & molecular biology [ 1545-9985 ] ; 2019.
English descriptors
- KwdEn :
- Bacteriophage lambda (genetics), CRISPR-Associated Protein 9 (metabolism), CRISPR-Cas Systems (genetics), Clustered Regularly Interspaced Short Palindromic Repeats (genetics), DNA Cleavage, DNA, Viral (genetics), DNA, Viral (metabolism), Escherichia coli (virology), Fluorescence Resonance Energy Transfer, Gene Editing, Microfluidics, Microscopy, Confocal, Optical Tweezers, RNA, Guide (genetics), Streptococcus pyogenes (enzymology).
- MESH :
- chemical , genetics : DNA, Viral, RNA, Guide.
- chemical , metabolism : CRISPR-Associated Protein 9, DNA, Viral.
- enzymology : Streptococcus pyogenes.
- genetics : Bacteriophage lambda, CRISPR-Cas Systems, Clustered Regularly Interspaced Short Palindromic Repeats.
- virology : Escherichia coli.
- DNA Cleavage, Fluorescence Resonance Energy Transfer, Gene Editing, Microfluidics, Microscopy, Confocal, Optical Tweezers.
Abstract
CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
DOI: 10.1038/s41594-019-0188-z
PubMed: 30804513
Links to Exploration step
pubmed:30804513Le document en format XML
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Driessen</name><affiliation><nlm:affiliation>LUMICKS BV, Amsterdam, The Netherlands.</nlm:affiliation></affiliation></author><author><name sortKey="Roos, Leonie" sort="Roos, Leonie" uniqKey="Roos L" first="Leonie" last="Roos">Leonie Roos</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Cvetesic, Nevena" sort="Cvetesic, Nevena" uniqKey="Cvetesic N" first="Nevena" last="Cvetesic">Nevena Cvetesic</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Allyjaun, Shenaz" sort="Allyjaun, Shenaz" uniqKey="Allyjaun S" first="Shenaz" last="Allyjaun">Shenaz Allyjaun</name><affiliation><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Lenhard, Boris" sort="Lenhard, Boris" uniqKey="Lenhard B" first="Boris" last="Lenhard">Boris Lenhard</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Cuomo, Maria Emanuela" sort="Cuomo, Maria Emanuela" uniqKey="Cuomo M" first="Maria Emanuela" last="Cuomo">Maria Emanuela Cuomo</name><affiliation><nlm:affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK. emanuela.cuomo@astrazeneca.com.</nlm:affiliation></affiliation></author><author><name sortKey="Rueda, David S" sort="Rueda, David S" uniqKey="Rueda D" first="David S" last="Rueda">David S. Rueda</name><affiliation><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK. david.rueda@imperial.ac.uk.</nlm:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PubMed</idno><date when="2019">2019</date><idno type="RBID">pubmed:30804513</idno><idno type="pmid">30804513</idno><idno type="doi">10.1038/s41594-019-0188-z</idno><idno type="wicri:Area/PubMed/Corpus">000928</idno><idno type="wicri:explorRef" wicri:stream="PubMed" wicri:step="Corpus" wicri:corpus="PubMed">000928</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en">DNA stretching induces Cas9 off-target activity.</title><author><name sortKey="Newton, Matthew D" sort="Newton, Matthew D" uniqKey="Newton M" first="Matthew D" last="Newton">Matthew D. Newton</name><affiliation><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Taylor, Benjamin J" sort="Taylor, Benjamin J" uniqKey="Taylor B" first="Benjamin J" last="Taylor">Benjamin J. Taylor</name><affiliation><nlm:affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Driessen, Rosalie P C" sort="Driessen, Rosalie P C" uniqKey="Driessen R" first="Rosalie P C" last="Driessen">Rosalie P C. Driessen</name><affiliation><nlm:affiliation>LUMICKS BV, Amsterdam, The Netherlands.</nlm:affiliation></affiliation></author><author><name sortKey="Roos, Leonie" sort="Roos, Leonie" uniqKey="Roos L" first="Leonie" last="Roos">Leonie Roos</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Cvetesic, Nevena" sort="Cvetesic, Nevena" uniqKey="Cvetesic N" first="Nevena" last="Cvetesic">Nevena Cvetesic</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Allyjaun, Shenaz" sort="Allyjaun, Shenaz" uniqKey="Allyjaun S" first="Shenaz" last="Allyjaun">Shenaz Allyjaun</name><affiliation><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Lenhard, Boris" sort="Lenhard, Boris" uniqKey="Lenhard B" first="Boris" last="Lenhard">Boris Lenhard</name><affiliation><nlm:affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</nlm:affiliation></affiliation></author><author><name sortKey="Cuomo, Maria Emanuela" sort="Cuomo, Maria Emanuela" uniqKey="Cuomo M" first="Maria Emanuela" last="Cuomo">Maria Emanuela Cuomo</name><affiliation><nlm:affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK. emanuela.cuomo@astrazeneca.com.</nlm:affiliation></affiliation></author><author><name sortKey="Rueda, David S" sort="Rueda, David S" uniqKey="Rueda D" first="David S" last="Rueda">David S. Rueda</name><affiliation><nlm:affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK. david.rueda@imperial.ac.uk.</nlm:affiliation></affiliation></author></analytic><series><title level="j">Nature structural & molecular biology</title><idno type="eISSN">1545-9985</idno><imprint><date when="2019" type="published">2019</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass><keywords scheme="KwdEn" xml:lang="en"><term>Bacteriophage lambda (genetics)</term><term>CRISPR-Associated Protein 9 (metabolism)</term><term>CRISPR-Cas Systems (genetics)</term><term>Clustered Regularly Interspaced Short Palindromic Repeats (genetics)</term><term>DNA Cleavage</term><term>DNA, Viral (genetics)</term><term>DNA, Viral (metabolism)</term><term>Escherichia coli (virology)</term><term>Fluorescence Resonance Energy Transfer</term><term>Gene Editing</term><term>Microfluidics</term><term>Microscopy, Confocal</term><term>Optical Tweezers</term><term>RNA, Guide (genetics)</term><term>Streptococcus pyogenes (enzymology)</term></keywords><keywords scheme="MESH" type="chemical" qualifier="genetics" xml:lang="en"><term>DNA, Viral</term><term>RNA, Guide</term></keywords><keywords scheme="MESH" type="chemical" qualifier="metabolism" xml:lang="en"><term>CRISPR-Associated Protein 9</term><term>DNA, Viral</term></keywords><keywords scheme="MESH" qualifier="enzymology" xml:lang="en"><term>Streptococcus pyogenes</term></keywords><keywords scheme="MESH" qualifier="genetics" xml:lang="en"><term>Bacteriophage lambda</term><term>CRISPR-Cas Systems</term><term>Clustered Regularly Interspaced Short Palindromic Repeats</term></keywords><keywords scheme="MESH" qualifier="virology" xml:lang="en"><term>Escherichia coli</term></keywords><keywords scheme="MESH" xml:lang="en"><term>DNA Cleavage</term><term>Fluorescence Resonance Energy Transfer</term><term>Gene Editing</term><term>Microfluidics</term><term>Microscopy, Confocal</term><term>Optical Tweezers</term></keywords></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en">CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.</div></front></TEI><pubmed><MedlineCitation Status="MEDLINE" Owner="NLM"><PMID Version="1">30804513</PMID><DateCompleted><Year>2019</Year><Month>11</Month><Day>27</Day></DateCompleted><DateRevised><Year>2020</Year><Month>03</Month><Day>09</Day></DateRevised><Article PubModel="Print-Electronic"><Journal><ISSN IssnType="Electronic">1545-9985</ISSN><JournalIssue CitedMedium="Internet"><Volume>26</Volume><Issue>3</Issue><PubDate><Year>2019</Year><Month>03</Month></PubDate></JournalIssue><Title>Nature structural & molecular biology</Title><ISOAbbreviation>Nat. Struct. Mol. Biol.</ISOAbbreviation></Journal><ArticleTitle>DNA stretching induces Cas9 off-target activity.</ArticleTitle><Pagination><MedlinePgn>185-192</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1038/s41594-019-0188-z</ELocationID><Abstract><AbstractText>CRISPR/Cas9 is a powerful genome-editing tool, but spurious off-target edits present a barrier to therapeutic applications. To understand how CRISPR/Cas9 discriminates between on-targets and off-targets, we have developed a single-molecule assay combining optical tweezers with fluorescence to monitor binding to λ-DNA. At low forces, the Streptococcus pyogenes Cas9 complex binds and cleaves DNA specifically. At higher forces, numerous off-target binding events appear repeatedly at the same off-target sites in a guide-RNA-sequence-dependent manner, driven by the mechanical distortion of the DNA. Using single-molecule Förster resonance energy transfer (smFRET) and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with ten mismatches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (for example, transcription, replication, etc.) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.</AbstractText></Abstract><AuthorList CompleteYN="Y"><Author ValidYN="Y"><LastName>Newton</LastName><ForeName>Matthew D</ForeName><Initials>MD</Initials><Identifier Source="ORCID">http://orcid.org/0000-0002-9453-6791</Identifier><AffiliationInfo><Affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Taylor</LastName><ForeName>Benjamin J</ForeName><Initials>BJ</Initials><Identifier Source="ORCID">http://orcid.org/0000-0001-6101-3786</Identifier><AffiliationInfo><Affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Driessen</LastName><ForeName>Rosalie P C</ForeName><Initials>RPC</Initials><AffiliationInfo><Affiliation>LUMICKS BV, Amsterdam, The Netherlands.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Roos</LastName><ForeName>Leonie</ForeName><Initials>L</Initials><AffiliationInfo><Affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cvetesic</LastName><ForeName>Nevena</ForeName><Initials>N</Initials><AffiliationInfo><Affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Allyjaun</LastName><ForeName>Shenaz</ForeName><Initials>S</Initials><AffiliationInfo><Affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Lenhard</LastName><ForeName>Boris</ForeName><Initials>B</Initials><Identifier Source="ORCID">http://orcid.org/0000-0002-1114-1509</Identifier><AffiliationInfo><Affiliation>Computational Regulatory Genomics, MRC-London Institute of Medical Sciences, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Institute of Clinical Sciences, Faculty of Medicine, Imperial College London, London, UK.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Sars International Centre for Marine Molecular Biology, University of Bergen, Bergen, Norway.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Cuomo</LastName><ForeName>Maria Emanuela</ForeName><Initials>ME</Initials><AffiliationInfo><Affiliation>Discovery Sciences, IMED Biotech Unit, Astra Zeneca, Cambridge, UK. emanuela.cuomo@astrazeneca.com.</Affiliation></AffiliationInfo></Author><Author ValidYN="Y"><LastName>Rueda</LastName><ForeName>David S</ForeName><Initials>DS</Initials><Identifier Source="ORCID">http://orcid.org/0000-0003-4657-6323</Identifier><AffiliationInfo><Affiliation>Molecular Virology, Department of Medicine, Imperial College London, London, UK. david.rueda@imperial.ac.uk.</Affiliation></AffiliationInfo><AffiliationInfo><Affiliation>Single Molecule Imaging Group, MRC-London Institute of Medical Sciences, London, UK. david.rueda@imperial.ac.uk.</Affiliation></AffiliationInfo></Author></AuthorList><Language>eng</Language><GrantList CompleteYN="Y"><Grant><GrantID>MC_UP_1102/1</GrantID><Acronym>MRC_</Acronym><Agency>Medical Research Council</Agency><Country>United Kingdom</Country></Grant><Grant><GrantID>MC_UP_1102/5</GrantID><Acronym>MRC_</Acronym><Agency>Medical Research Council</Agency><Country>United Kingdom</Country></Grant><Grant><GrantID>106954</GrantID><Acronym>WT_</Acronym><Agency>Wellcome Trust</Agency><Country>United Kingdom</Country></Grant><Grant><GrantID>206292/Z/17/Z</GrantID><Acronym>WT_</Acronym><Agency>Wellcome Trust</Agency><Country>United Kingdom</Country></Grant></GrantList><PublicationTypeList><PublicationType UI="D016428">Journal Article</PublicationType><PublicationType UI="D013485">Research Support, Non-U.S. Gov't</PublicationType></PublicationTypeList><ArticleDate DateType="Electronic"><Year>2019</Year><Month>02</Month><Day>25</Day></ArticleDate></Article><MedlineJournalInfo><Country>United States</Country><MedlineTA>Nat Struct Mol Biol</MedlineTA><NlmUniqueID>101186374</NlmUniqueID><ISSNLinking>1545-9985</ISSNLinking></MedlineJournalInfo><ChemicalList><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D004279">DNA, Viral</NameOfSubstance></Chemical><Chemical><RegistryNumber>0</RegistryNumber><NameOfSubstance UI="D017394">RNA, Guide</NameOfSubstance></Chemical><Chemical><RegistryNumber>EC 3.1.-</RegistryNumber><NameOfSubstance UI="D000076987">CRISPR-Associated Protein 9</NameOfSubstance></Chemical></ChemicalList><CitationSubset>IM</CitationSubset><MeshHeadingList><MeshHeading><DescriptorName UI="D010582" MajorTopicYN="N">Bacteriophage lambda</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D000076987" MajorTopicYN="N">CRISPR-Associated Protein 9</DescriptorName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D064113" MajorTopicYN="N">CRISPR-Cas Systems</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D064112" MajorTopicYN="N">Clustered Regularly Interspaced Short Palindromic Repeats</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="Y">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D053837" MajorTopicYN="N">DNA Cleavage</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D004279" MajorTopicYN="N">DNA, Viral</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName><QualifierName UI="Q000378" MajorTopicYN="Y">metabolism</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D004926" MajorTopicYN="N">Escherichia coli</DescriptorName><QualifierName UI="Q000821" MajorTopicYN="N">virology</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D031541" MajorTopicYN="N">Fluorescence Resonance Energy Transfer</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D000072669" MajorTopicYN="N">Gene Editing</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D044085" MajorTopicYN="N">Microfluidics</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D018613" MajorTopicYN="N">Microscopy, Confocal</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D052898" MajorTopicYN="N">Optical Tweezers</DescriptorName></MeshHeading><MeshHeading><DescriptorName UI="D017394" MajorTopicYN="N">RNA, Guide</DescriptorName><QualifierName UI="Q000235" MajorTopicYN="N">genetics</QualifierName></MeshHeading><MeshHeading><DescriptorName UI="D013297" MajorTopicYN="N">Streptococcus pyogenes</DescriptorName><QualifierName UI="Q000201" MajorTopicYN="N">enzymology</QualifierName></MeshHeading></MeshHeadingList></MedlineCitation><PubmedData><History><PubMedPubDate PubStatus="received"><Year>2018</Year><Month>11</Month><Day>02</Day></PubMedPubDate><PubMedPubDate PubStatus="accepted"><Year>2019</Year><Month>01</Month><Day>18</Day></PubMedPubDate><PubMedPubDate PubStatus="pubmed"><Year>2019</Year><Month>2</Month><Day>26</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="medline"><Year>2019</Year><Month>11</Month><Day>28</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate><PubMedPubDate PubStatus="entrez"><Year>2019</Year><Month>2</Month><Day>27</Day><Hour>6</Hour><Minute>0</Minute></PubMedPubDate></History><PublicationStatus>ppublish</PublicationStatus><ArticleIdList><ArticleId IdType="pubmed">30804513</ArticleId><ArticleId IdType="doi">10.1038/s41594-019-0188-z</ArticleId><ArticleId IdType="pii">10.1038/s41594-019-0188-z</ArticleId></ArticleIdList></PubmedData></pubmed></record>Pour manipuler 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