RT Journal Article
SR Electronic
T1 Pairwise library screen systematically interrogates <em>Staphylococcus aureus</em> Cas9 specificity in human cells
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 269399
DO 10.1101/269399
A1 Tycko, Josh
A1 Barrera, Luis A.
A1 Huston, Nicholas
A1 Friedland, Ari E.
A1 Wu, Xuebing
A1 Gootenberg, Jonathan S.
A1 Abudayyeh, Omar O.
A1 Myer, Vic E.
A1 Wilson, Christopher J.
A1 Hsu, Patrick D.
YR 2018
UL http://biorxiv.org/content/early/2018/02/22/269399.abstract
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.