4.8 Article

Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells

Journal

NATURE BIOTECHNOLOGY
Volume 32, Issue 7, Pages 670-+

Publisher

NATURE PUBLISHING GROUP
DOI: 10.1038/nbt.2889

Keywords

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Funding

  1. United States Public Health Service from the National Institutes of Health [RO1-GM34277, R01-CA133404]
  2. National Cancer Institute [PO1-CA42063]
  3. Cancer Center Support (core) grant from the National Cancer Institute [P30-CA14051]
  4. US National Institutes of Health Director's Pioneer Award [1DP1-MH100706]
  5. Keck Foundation
  6. McKnight Foundation
  7. Poitras Foundation
  8. Merkin Foundation
  9. Vallee Foundation
  10. Damon Runyon Foundation
  11. Searle Scholars Foundation
  12. Klingenstein Foundation
  13. Simons Foundation

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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.

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