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- Title
Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs.
- Authors
Xu, Ping; Liu, Zhiheng; Liu, Ying; Ma, Huazheng; Xu, Yiyuan; Bao, Ying; Zhu, Shiyou; Cao, Zhongzheng; Wu, Zeguang; Zhou, Zhuo; Wei, Wensheng
- Abstract
Canonical CRISPR–knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR–KO screens in various settings. CRISPR genetic screens with base editors avoid the confounding effects of DNA breaks.
- Publication
Nature Biotechnology, 2021, Vol 39, Issue 11, p1403
- ISSN
1087-0156
- Publication type
Article
- DOI
10.1038/s41587-021-00944-1