title: GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.
abstract: CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.|Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Zongli
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Nhu T
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Matthew
authors.initials: M
authors.lastname: Liebers
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Ved V
authors.initials: VV
authors.lastname: Topkar
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Vishal
authors.initials: V
authors.lastname: Thapar
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Wyvekens
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.
authors.forename: Cyd
authors.initials: C
authors.lastname: Khayter
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.
authors.forename: A John
authors.initials: AJ
authors.lastname: Iafrate
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.
authors.forename: Long P
authors.initials: LP
authors.lastname: Le
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.
authors.forename: Martin J
authors.initials: MJ
authors.lastname: Aryee
authors.affiliation: Molecular Pathology Unit, Massachusetts General Hospital, Charlestown, MA USA.|Center for Cancer Research, Massachusetts General Hospital, Charlestown, MA USA.|Department of Pathology, Harvard Medical School, Boston, MA USA.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D064113:CRISPR-Cas Systems / Q000235:genetics*; D002460:Cell Line; D053903:DNA Breaks, Double-Stranded; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009838:Oligodeoxyribonucleotides / Q000235:genetics; D017393:RNA Editing / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics*
chemical_list: D009838:Oligodeoxyribonucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems