In silico abstraction of zinc finger nuclease cleavage profiles reveals an expanded landscape of off-target sites.
Gene-editing nucleases enable targeted modification of DNA sequences in living cells, thereby facilitating efficient knockout and precise editing of endogenous loci. Engineered nucleases also have the potential to introduce mutations at off-target sites of action. Such unintended alterations can confound interpretation of experiments and can have implications for development of therapeutic applications. Recently, two improved methods for identifying the off-target effects of zinc finger nucleases (ZFNs) were described-one using an in vitro cleavage site selection method and the other exploiting the insertion of integration-defective lentiviruses into nuclease-induced double-stranded DNA breaks. However, application of these two methods to a ZFN pair targeted to the human CCR5 gene led to identification of largely non-overlapping off-target sites, raising the possibility that additional off-target sites might exist. Here, we show that in silico abstraction of ZFN cleavage profiles obtained from in vitro cleavage site selections can greatly enhance the ability to identify potential off-target sites in human cells. Our improved method should enable more comprehensive profiling of ZFN specificities.
Molecular Pathology Unit, Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA 02129, USA, Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, MA 02129, USA, Department of Pathology, Harvard Medical School, Boston, MA 02115, USA, Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA 02115, USA, Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 01238, USA, Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA and Howard Hughes Medical Institute, Chevy Chase, MD 02815, USA.
Jeffry D
JD
Sander
Cherie L
CL
Ramirez
Samantha J
SJ
Linder
Vikram
V
Pattanayak
Noam
N
Shoresh
Manching
M
Ku
Jennifer A
JA
Foden
Deepak
D
Reyon
Bradley E
BE
Bernstein
David R
DR
Liu
J Keith
JK
Joung
D001185:Artificial Intelligence; D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000378:metabolism*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D019713:Receptors, CCR5 / Q000235:genetics; D017422:Sequence Analysis, DNA; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D016335:Zinc Fingers
D019713:Receptors, CCR5; C467484:VEGFA protein, human; D042461:Vascular Endothelial Growth Factor A; D004247:DNA; D003851:Deoxyribonucleases