Unknown

Dataset Information

0

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


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.

SUBMITTER: Tycko J 

PROVIDER: S-EPMC6063963 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

altmetric image

Publications

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

Tycko Josh J   Barrera Luis A LA   Huston Nicholas C NC   Friedland Ari E AE   Wu Xuebing X   Gootenberg Jonathan S JS   Abudayyeh Omar O OO   Myer Vic E VE   Wilson Christopher J CJ   Hsu Patrick D PD  

Nature communications 20180727 1


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  ...[more]

Similar Datasets

| S-EPMC6800346 | biostudies-literature
| S-EPMC3032424 | biostudies-literature
| S-EPMC4790305 | biostudies-literature
| S-EPMC4670267 | biostudies-literature
| S-EPMC6800334 | biostudies-literature
| S-EPMC6004052 | biostudies-literature
| S-EPMC9979987 | biostudies-literature
| S-EPMC4393360 | biostudies-literature
2023-07-11 | GSE211073 | GEO
| S-EPMC8216286 | biostudies-literature