Canonical CRISPR screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene knockouts. These DSB-dependent methodologies may yield false-positive results by mistakenly assuming targeted loci as essential for cell viability, especially when high-copy-number sites are targeted. Here, we use CRISPR cytosine base editors for genome-scale knockout screens by perturbing gene start codons or splice sites, or by introducing premature termination codons (PTCs). Combining with iBAR strategy we have previously established, we realized an iBARed cytosine Base Editing-mediated gene KnockOut (BARBEKO) screening strategy at a genome-scale (targeting 17,501 genes) in multiple human cell lines. By constructing such a cell library through lentiviral infection at a high MOI (up to 10), we significantly reduced starting cells while producing screening results with improved efficiency and accuracy. More importantly, in comparison with Cas9-mediated cell fitness screens, BARBEKO screens are no longer affected by DNA-cleavage induced cytotoxicity in HeLa, K562, or DSB-sensitive RPE1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR screens in various settings.