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

Author

Listed:
  • Josh Tycko

    (Editas Medicine
    Stanford University School of Medicine)

  • Luis A. Barrera

    (Editas Medicine
    Arrakis Therapeutics)

  • Nicholas C. Huston

    (Editas Medicine
    Yale University)

  • Ari E. Friedland

    (Editas Medicine)

  • Xuebing Wu

    (Whitehead Institute for Biomedical Research)

  • Jonathan S. Gootenberg

    (Harvard)

  • Omar O. Abudayyeh

    (Massachusetts Institute of Technology)

  • Vic E. Myer

    (Editas Medicine)

  • Christopher J. Wilson

    (Editas Medicine)

  • Patrick D. Hsu

    (Editas Medicine
    Salk Institute for Biological Studies)

Abstract

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.

Suggested Citation

  • Josh Tycko & Luis A. Barrera & Nicholas C. Huston & Ari E. Friedland & Xuebing Wu & Jonathan S. Gootenberg & Omar O. Abudayyeh & Vic E. Myer & Christopher J. Wilson & Patrick D. Hsu, 2018. "Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells," Nature Communications, Nature, vol. 9(1), pages 1-7, December.
    • Handle: RePEc:nat:natcom:v:9:y:2018:i:1:d:10.1038_s41467-018-05391-2
    DOI: 10.1038/s41467-018-05391-2
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    Cited by:

    1. Chengdong Zhang & Yuan Yang & Tao Qi & Yuening Zhang & Linghui Hou & Jingjing Wei & Jingcheng Yang & Leming Shi & Sang-Ging Ong & Hongyan Wang & Hui Wang & Bo Yu & Yongming Wang, 2023. "Prediction of base editor off-targets by deep learning," Nature Communications, Nature, vol. 14(1), pages 1-11, December.
    2. Nathan Bamidele & Han Zhang & Xiaolong Dong & Haoyang Cheng & Nicholas Gaston & Hailey Feinzig & Hanbing Cao & Karen Kelly & Jonathan K. Watts & Jun Xie & Guangping Gao & Erik J. Sontheimer, 2024. "Domain-inlaid Nme2Cas9 adenine base editors with improved activity and targeting scope," Nature Communications, Nature, vol. 15(1), pages 1-13, December.

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