Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease.
RNA-guided genome editing with the CRISPR-Cas9 system has great potential for basic and clinical research, but the determinants of targeting specificity and the extent of off-target cleavage remain insufficiently understood. Using chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), we mapped genome-wide binding sites of catalytically inactive Cas9 (dCas9) in HEK293T cells, in combination with 12 different single guide RNAs (sgRNAs). The number of off-target sites bound by dCas9 varied from ∼10 to >1,000 depending on the sgRNA. Analysis of off-target binding sites showed the importance of the PAM-proximal region of the sgRNA guiding sequence and that dCas9 binding sites are enriched in open chromatin regions. When targeted with catalytically active Cas9, some off-target binding sites had indels above background levels in a region around the ChIP-seq peak, but generally at lower rates than the on-target sites. Our results elucidate major determinants of Cas9 targeting, and we show that ChIP-seq allows unbiased detection of Cas9 binding sites genome-wide.
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2].
Cem
C
Kuscu
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2] Department of Biology, Pamukkale University, Denizli, Turkey. [3].
Sevki
S
Arslan
1] School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA. [2] Department of Computer Science, University of Virginia, Charlottesville, Virginia, USA.
Ritambhara
R
Singh
School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.
Jeremy
J
Thorpe
School of Medicine, Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, Virginia, USA.
Mazhar
M
Adli
D001483:Base Sequence; D001665:Binding Sites; D064113:CRISPR-Cas Systems / Q000235:genetics*; D002478:Cells, Cultured; D002874:Chromosome Mapping; D003850:Deoxyribonuclease I / Q000235:genetics*; D053595:Embryonic Stem Cells / Q000502:physiology*; D018390:Gene Targeting / Q000379:methods*; D016678:Genome / Q000235:genetics*; D057809:HEK293 Cells; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data
D003850:Deoxyribonuclease I