Imperial College London

ProfessorBorisLenhard

Faculty of MedicineInstitute of Clinical Sciences

Professor of Computational Biology
 
 
 
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Contact

 

+44 (0)20 3313 8353b.lenhard Website

 
 
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Assistant

 

Mr Alastair Douglas Ivor Williams +44 (0)20 3313 4318

 
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Location

 

230ICTEM buildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@article{Newton:2019:10.1038/s41594-019-0188-z,
author = {Newton, MD and Taylor, BJ and Driessen, RPC and Roos, L and Cvetesic, N and Allyjaun, S and Lenhard, B and Cuomo, ME and Rueda, DS},
doi = {10.1038/s41594-019-0188-z},
journal = {Nature Structural and Molecular Biology},
pages = {185--192},
title = {DNA stretching induces Cas9 off-target activity},
url = {http://dx.doi.org/10.1038/s41594-019-0188-z},
volume = {26},
year = {2019}
}

RIS format (EndNote, RefMan)

TY  - JOUR
AB - 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.
AU - Newton,MD
AU - Taylor,BJ
AU - Driessen,RPC
AU - Roos,L
AU - Cvetesic,N
AU - Allyjaun,S
AU - Lenhard,B
AU - Cuomo,ME
AU - Rueda,DS
DO - 10.1038/s41594-019-0188-z
EP - 192
PY - 2019///
SN - 1545-9985
SP - 185
TI - DNA stretching induces Cas9 off-target activity
T2 - Nature Structural and Molecular Biology
UR - http://dx.doi.org/10.1038/s41594-019-0188-z
UR - http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000460284300007&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
UR - http://hdl.handle.net/10044/1/70379
VL - 26
ER -