Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells

Nat Commun. 2018 Jul 27;9(1):2962. doi: 10.1038/s41467-018-05391-2.


Therapeutic genome editing with Staphylococcus aureus Cas9 (SaCas9) requires a rigorous understanding of its potential off-target activity in the human genome. Here we report a high-throughput screening approach to measure SaCas9 genome editing variation in human cells across a large repertoire of 88,692 single guide RNAs (sgRNAs) paired with matched or mismatched target sites in a synthetic cassette. We incorporate randomized barcodes that enable whitelisting of correctly synthesized molecules for further downstream analysis, in order to circumvent the limitation of oligonucleotide synthesis errors. We find SaCas9 sgRNAs with 21-mer or 22-mer spacer sequences are generally more active, although high efficiency 20-mer spacers are markedly less tolerant of mismatches. Using this dataset, we developed an SaCas9 specificity model that performs robustly in ranking off-target sites. The barcoded pairwise library screen enabled high-fidelity recovery of guide-target relationships, providing a scalable framework for the investigation of CRISPR enzyme properties and general nucleic acid interactions.

MeSH terms

  • Bacterial Proteins / genetics
  • Base Sequence
  • CRISPR-Associated Protein 9 / genetics*
  • CRISPR-Cas Systems
  • Cloning, Molecular
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Gene Editing / methods*
  • Gene Library*
  • Genes, Bacterial / genetics
  • HEK293 Cells
  • Humans
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Staphylococcus aureus / genetics*


  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9