Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells
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Jaenisch_Genome-wide.pdf
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Author(s)
Wu, Xuebing
Cheng, Albert W.
Chen, Sidi
Jaenisch, Rudolf
Zhang, Feng
Chiu, Anthony Chun-yin
Sharp, Phillip A.
Scott, David Arthur
Hsu, Patrick
Trevino, Alexandro E.
Date Issued
April 2014
Journal
Nature Biotechnology
Publisher
Nature Publishing Group
Citation
Wu, Xuebing, David A Scott, Andrea J Kriz, Anthony C Chiu, Patrick D Hsu, Daniel B Dadon, Albert W Cheng, et al. “Genome-Wide Binding of the CRISPR Endonuclease Cas9 in Mammalian Cells.” Nature Biotechnology 32, no. 7 (April 20, 2014): 670–676.
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Author's final manuscript
Abstract
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.
MIT Department
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
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DOI of Published Version
https://doi.org/10.1038/nbt.2889
