PT  - JOURNAL ARTICLE
AU  - Josh Tycko
AU  - Luis A. Barrera
AU  - Nicholas Huston
AU  - Ari E. Friedland
AU  - Xuebing Wu
AU  - Jonathan S. Gootenberg
AU  - Omar O. Abudayyeh
AU  - Vic E. Myer
AU  - Christopher J. Wilson
AU  - Patrick D. Hsu
TI  - Pairwise library screen systematically interrogates <em>Staphylococcus aureus</em> Cas9 specificity in human cells
AID  - 10.1101/269399
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 269399
4099  - http://biorxiv.org/content/early/2018/02/22/269399.short
4100  - http://biorxiv.org/content/early/2018/02/22/269399.full
AB  - We report a high-throughput screening approach to measure Staphylococcus aureus Cas9 (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 incorporated 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 a 21-nucleotide spacer are most active against off-targets with single and double mismatches, compared to shorter or longer sgRNAs. Using this dataset, we developed an SaCas9 specificity model that performs well 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.