Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

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.

Automated summary

BARBEKO is a DSB-independent, genome-wide CRISPR screening method that leverages CRISPR cytosine base editors to perturb gene start codons or splice sites for KO screening. By integrating with the iBAR strategy, BARBEKO improves screening quality and efficiency, yielding efficient and accurate results with reduced starting cells. Moreover, BARBEKO screens are not impacted by DNA cleavage-induced cytotoxicity in specific cell types, offering a valuable tool to complement current CRISPR-KO screens.