Imperial College London
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ProfessorBorisLenhard

Faculty of Medicine, Institute of Clinical Sciences

Professor of Computational Biology
 
 
 
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Contact

 

+44 (0)20 7594 0911b.lenhard Website

 
 
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Assistant

 

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

 
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Location

 

6.12CLMS BuildingHammersmith Campus

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Summary

 

Publications

Citation

BibTex format

@article{Newton:2018,
author = {Newton, M and Taylor, B and Driessen, R and Roos, L and Cvetesic, N and Allyjaun, S and Lenhard, B and Cuomo, ME and Rueda, D},
journal = {Nature Structural and Molecular Biology},
title = {DNA stretching induces Cas9 off-target binding and cleavage},
url = {http://hdl.handle.net/10044/1/66907},
year = {2018}
}
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RIS format (EndNote, RefMan)

TY  - JOUR
AB  - CRISPR/Cas9 is a powerful genome editing tool, but spurious off-target edits present a barrier towards therapeutic applications. To understand how CRISPR/Cas9 discrimi-nates between on- and off-targets, we have developed a single-molecule assay com-bining 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 FRET and cleavage assays, we show that DNA bubbles induce off-target binding and cleavage at these sites, even with 10 mis-matches, as well as at previously identified in vivo off-targets. We propose that duplex DNA destabilization during cellular processes (e.g., transcription, replication, etc) can expose these cryptic off-target sites to Cas9 activity, highlighting the need for improved off-target prediction algorithms.
AU  - Newton,M
AU  - Taylor,B
AU  - Driessen,R
AU  - Roos,L
AU  - Cvetesic,N
AU  - Allyjaun,S
AU  - Lenhard,B
AU  - Cuomo,ME
AU  - Rueda,D
PY  - 2018///
SN  - 1545-9985
TI  - DNA stretching induces Cas9 off-target binding and cleavage
T2  - Nature Structural and Molecular Biology
UR  - http://hdl.handle.net/10044/1/66907
ER  -
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