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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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