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Billon, Pierre; Bryant, Eric E.; Joseph, Sarah A.; Nambiar, Tarun S.; Hayward, Samuel B.; Rothstein, Rodney; Ciccia, Alberto
Molecular cell, 09/2017, Letnik: 67, Številka: 6Journal Article
Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%–99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP. Display omitted •CRISPR-mediated base editing allows the conversion of four codons into STOP codons•Induction of STOP codons (iSTOP) can be monitored using restriction enzymes•97%–99% of genes in eight eukaryotic species are targetable by iSTOP•iSTOP can be employed to model over 32,000 cancer-associated nonsense mutations Billon et al. describe a CRISPR-based approach to inactivate genes by directly converting four codons into STOP codons. The induction of STOP codons (iSTOP) can be rapidly monitored using restriction enzymes. iSTOP can be employed to inactivate eukaryotic genes on a genome-wide scale and model cancer-associated nonsense mutations.
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