Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
| العنوان: | Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells |
|---|---|
| المؤلفون: | Alexandro E. Trevino, Anthony C. Chiu, Patrick D. Hsu, David A. Scott, Sidi Chen, Albert W. Cheng, Andrea J. Kriz, Phillip A. Sharp, Silvana Konermann, Feng Zhang, Daniel B. Dadon, Rudolf Jaenisch, Xuebing Wu |
| المساهمون: | Massachusetts Institute of Technology. Department of Biology, McGovern Institute for Brain Research at MIT, Whitehead Institute for Biomedical Research, Koch Institute for Integrative Cancer Research at MIT, Wu, Xuebing, Chiu, Anthony Chun-yin, Chen, Sidi, Sharp, Phillip A., Scott, David Arthur, Hsu, Patrick, Trevino, Alexandro E., Konermann, Silvana M., Zhang, Feng, Kriz, Andrea J., Dadon, Daniel Benjamin, Jaenisch, Rudolf |
| المصدر: | Nature biotechnology, vol 32, iss 7 PMC |
| سنة النشر: | 2014 |
| مصطلحات موضوعية: | Cells, Molecular Sequence Data, Biomedical Engineering, Bioengineering, Biology, Applied Microbiology and Biotechnology, Article, Mice, Genetic, Models, CRISPR, Animals, Deoxyribonuclease I, Guide RNA, Binding site, Gene, Cells, Cultured, Embryonic Stem Cells, Genetics, Cultured, Binding Sites, Genome, Base Sequence, Models, Genetic, Cas9, Chromatin, DNA-Binding Proteins, Protospacer adjacent motif, Molecular Medicine, CRISPR-Cas Systems, Biotechnology, Protein Binding |
| الوصف: | Bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood. Here we mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). Each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. Targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. We propose a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage. National Institutes of Health (U.S.) (Grant RO1-GM34277) National Institutes of Health (U.S.) (Grant R01-CA133404) National Cancer Institute (U.S.) (Grant PO1-CA42063) National Cancer Institute (U.S.) (Cancer Center Support (Core) Grant P30-CA14051) National Institutes of Health (U.S.) (Director's Pioneer Award 1DP1-MH100706) Damon Runyon Cancer Research Foundation Kinship Foundation. Searle Scholars Program Simons Foundation |
| وصف الملف: | application/pdf |
| اللغة: | English |
| الوصول الحر: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::d93a41c1c755061d128c6bddf0d92a93Test https://europepmc.org/articles/PMC4145672Test/ |
| حقوق: | OPEN |
| رقم الانضمام: | edsair.doi.dedup.....d93a41c1c755061d128c6bddf0d92a93 |
| قاعدة البيانات: | OpenAIRE |
| الوصف غير متاح. |