Imperial College London


Faculty of MedicineInstitute of Clinical Sciences

Professor of Computational Biology



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




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




230ICTEM buildingHammersmith Campus






BibTex format

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 = {},

RIS format (EndNote, RefMan)

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
SN - 1545-9985
TI - DNA stretching induces Cas9 off-target binding and cleavage
T2 - Nature Structural and Molecular Biology
UR -
ER -