Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease

Cem Kuscu, Sevki Arslan, Ritambhara Singh, Jeremy Thorpe, Mazhar Adli

Research output: Contribution to journalArticle

357 Citations (Scopus)

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.

Original languageEnglish (US)
Pages (from-to)677-683
Number of pages7
JournalNature Biotechnology
Volume32
Issue number7
DOIs
StatePublished - Jan 1 2014

Fingerprint

Endonucleases
Binding sites
Genes
Binding Sites
Chromatin
Genome
Chromatin Immunoprecipitation
Throughput
RNA
Clustered Regularly Interspaced Short Palindromic Repeats
Guide RNA
Pulse amplitude modulation
Research

All Science Journal Classification (ASJC) codes

  • Applied Microbiology and Biotechnology
  • Biotechnology
  • Molecular Medicine
  • Bioengineering
  • Biomedical Engineering

Cite this

Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. / Kuscu, Cem; Arslan, Sevki; Singh, Ritambhara; Thorpe, Jeremy; Adli, Mazhar.

In: Nature Biotechnology, Vol. 32, No. 7, 01.01.2014, p. 677-683.

Research output: Contribution to journalArticle

Kuscu, Cem ; Arslan, Sevki ; Singh, Ritambhara ; Thorpe, Jeremy ; Adli, Mazhar. / Genome-wide analysis reveals characteristics of off-target sites bound by the Cas9 endonuclease. In: Nature Biotechnology. 2014 ; Vol. 32, No. 7. pp. 677-683.
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