Please use this identifier to cite or link to this item: https://hdl.handle.net/11499/7681
Title: Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease
Authors: Kuscu, C.
Arslan, Şevki
Singh, R.
Thorpe, J.
Adli, M.
Keywords: Nucleic acids
Background level
Chromatin immunoprecipitation
Clinical research
Endonucleases
High-throughput sequencing
Genes
cas9 endonuclease
endonuclease
guide RNA
unclassified drug
article
binding site
catalysis
chromatin
chromatin immunoprecipitation
controlled study
embryo
enzyme activity
enzyme binding
genome analysis
HEK293 cell line
high throughput sequencing
human
human cell
indel mutation
nucleotide sequence
priority journal
protein cleavage
protein RNA binding
RNA sequence
Base Sequence
Binding Sites
Cells, Cultured
Chromosome Mapping
CRISPR-Cas Systems
Deoxyribonuclease I
Embryonic Stem Cells
Gene Targeting
Genome
HEK293 Cells
Humans
Models, Genetic
Molecular Sequence Data
Publisher: Nature Publishing Group
Abstract: 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 â ^1/410 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. © 2014 Nature America, Inc.
URI: https://hdl.handle.net/11499/7681
https://doi.org/10.1038/nbt.2916
ISSN: 1087-0156
Appears in Collections:Fen-Edebiyat Fakültesi Koleksiyonu
PubMed İndeksli Yayınlar Koleksiyonu / PubMed Indexed Publications Collection
Scopus İndeksli Yayınlar Koleksiyonu / Scopus Indexed Publications Collection
WoS İndeksli Yayınlar Koleksiyonu / WoS Indexed Publications Collection

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