Pairwise library screen systematically interrogates Staphylococcus aureus Cas9 specificity in human cells
| Autor: | Luis A. Barrera, Josh Tycko, Patrick D. Hsu, Jonathan S. Gootenberg, Ari E. Friedland, Christine D. Wilson, Xuebing Wu, Vic E. Myer, Omar O. Abudayyeh, Nicholas C. Huston |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Staphylococcus aureus Science General Physics and Astronomy Computational biology Oligonucleotide synthesis Biology medicine.disease_cause Article General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Synthetic biology Bacterial Proteins Genome editing CRISPR-Associated Protein 9 Genetics medicine Humans CRISPR Clustered Regularly Interspaced Short Palindromic Repeats Genomic library Guide RNA lcsh:Science Gene Gene Library Gene Editing Multidisciplinary Base Sequence Cas9 Human Genome Bacterial Molecular General Chemistry HEK293 Cells 030104 developmental biology Genes Nucleic acid RNA Pairwise comparison lcsh:Q Human genome CRISPR-Cas Systems Guide Cloning Biotechnology |
| Zdroj: | Nature Communications, Vol 9, Iss 1, Pp 1-7 (2018) Nature Communications Nature communications, vol 9, iss 1 |
| ISSN: | 2041-1723 |
| Popis: | 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. A rigorous understanding of off-target effects is necessary for SaCas9 to be used in therapeutic genome editing. Here the authors measure SaCas9 mismatch tolerance across a pairwise library screen of 88,000 guides and targets in human cells and develop a model which ranks off-target sites. |
| Databáze: | OpenAIRE |
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