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, 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 = {},
volume = {26},
year = {2019}

RIS format (EndNote, RefMan)

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 -
UR -
UR -
VL - 26
ER -