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

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

Author information

1
Editas Medicine, 11 Hurley St., Cambridge, MA, 02141, USA.
2
Department of Genetics, Stanford University School of Medicine, Stanford, CA, 94305, USA.
3
Arrakis Therapeutics, 35 Gatehouse Dr., Waltham, MA, 02451, USA.
4
Department of Molecular Biophysics and Biochemistry, Yale University, New Haven, CT, 06511, USA.
5
Whitehead Institute for Biomedical Research, Cambridge, MA, 02142, USA.
6
Department of Systems Biology, Harvard, Cambridge, MA, 02138, USA.
7
Department of Health Sciences and Technology, Massachusetts Institute of Technology, Cambridge, MA, 02139, USA.
8
Editas Medicine, 11 Hurley St., Cambridge, MA, 02141, USA. christopher.wilson@editasmed.com.
9
Editas Medicine, 11 Hurley St., Cambridge, MA, 02141, USA. patrick@salk.edu.
10
Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, La Jolla, CA, 92037, USA. patrick@salk.edu.

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.

PMID:
30054474
PMCID:
PMC6063963
DOI:
10.1038/s41467-018-05391-2
[Indexed for MEDLINE]
Free PMC Article

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