Mapping the genomic landscape of CRISPR-Cas9 cleavage

Nat Methods. 2017 Jun;14(6):600-606. doi: 10.1038/nmeth.4284. Epub 2017 May 1.


RNA-guided CRISPR-Cas9 endonucleases are widely used for genome engineering, but our understanding of Cas9 specificity remains incomplete. Here, we developed a biochemical method (SITE-Seq), using Cas9 programmed with single-guide RNAs (sgRNAs), to identify the sequence of cut sites within genomic DNA. Cells edited with the same Cas9-sgRNA complexes are then assayed for mutations at each cut site using amplicon sequencing. We used SITE-Seq to examine Cas9 specificity with sgRNAs targeting the human genome. The number of sites identified depended on sgRNA sequence and nuclease concentration. Sites identified at lower concentrations showed a higher propensity for off-target mutations in cells. The list of off-target sites showing activity in cells was influenced by sgRNP delivery, cell type and duration of exposure to the nuclease. Collectively, our results underscore the utility of combining comprehensive biochemical identification of off-target sites with independent cell-based measurements of activity at those sites when assessing nuclease activity and specificity.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Chromosome Mapping / methods*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Genome / genetics*
  • High-Throughput Nucleotide Sequencing
  • Sequence Analysis, DNA