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

Details

• Alternative Title:
  Nat Biotechnol
• Personal Author:
  Wu, Xuebing ; Scott, David A. ; Kriz, Andrea J. ; Chiu, Anthony C. ; Hsu, Patrick D. ; Dadon, Daniel B. ; Cheng, Albert W. ; Trevino, Alexandro E. ; Konermann, Silvana ; Chen, Sidi ; Jaenisch, Rudolf ; Zhang, Feng ; Sharp, Phillip A.
• Description:
  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.
• Keywords:
  Animals Base Sequence Binding Sites Cells, Cultured CRISPR-Cas Systems Deoxyribonuclease I DNA-Binding Proteins Embryonic Stem Cells Genome Mice Models, Genetic Molecular Sequence Data Protein Binding

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• Source:
  Nat Biotechnol. 32(7):670-676
• Pubmed ID:
  24752079
• Pubmed Central ID:
  PMC4145672
• Document Type:
  Journal Article
• Funding:
  T32 GM007287/GM/NIGMS NIH HHSUnited States/ ; R01-CA133404/CA/NCI NIH HHSUnited States/ ; P30 CA014051/CA/NCI NIH HHSUnited States/ ; P30-CA14051/CA/NCI NIH HHSUnited States/ ; R37 HD045022/HD/NICHD NIH HHSUnited States/ ; R01 GM034277/GM/NIGMS NIH HHSUnited States/ ; R01 DK097768/DK/NIDDK NIH HHSUnited States/ ; P01-CA42063/CA/NCI NIH HHSUnited States/ ; 1DP1-MH100706/DP/NCCDPHP CDC HHSUnited States/ ; R01-GM34277/GM/NIGMS NIH HHSUnited States/ ; HHMI/Howard Hughes Medical InstituteUnited States/ ; P01 CA042063/CA/NCI NIH HHSUnited States/ ; R37 GM034277/GM/NIGMS NIH HHSUnited States/ ; R01 CA133404/CA/NCI NIH HHSUnited States/ ; DP1 MH100706/MH/NIMH NIH HHSUnited States/
• Volume:
  32
• Issue:
  7
• Collection(s):
  CDC Public Access
• Main Document Checksum:
  urn:sha256:ff895aee20075048d256d9b63357bbdf8b884df465e9bcfc10d51679240bfef7
• Download URL:
  https://stacks.cdc.gov/view/cdc/30092/cdc_30092_DS1.pdf
• File Type:
  [PDF - 1.31 MB ]