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

Xu, P;Liu, Z;Liu, Y;Ma, H;Xu, Y;Bao, Y;Zhu, S;Cao, Z;Zhou, Z;Wei, W;

Canonical CRISPR screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate 18 targeted gene knockouts. These DSB-dependent methodologies may yield false-positive results by 19 mistakenly assuming targeted loci as essential for cell viability, especially when high-copy-number 20 sites are targeted. Here, we use CRISPR cytosine base editors for genome-scale knockout screens by 21 perturbing gene start codons or splice sites, or by introducing premature termination codons (PTCs). 22 Combining with iBAR strategy we have previously established, we realized an iBARed cytosine Base 23 Editing-mediated gene KnockOut (BARBEKO) screening strategy at a genome-scale (targeting 17,501 24 genes) in multiple human cell lines. By constructing such a cell library through lentiviral infection at 25 a high MOI (up to 10), we significantly reduced starting cells while producing screening results with 26 improved efficiency and accuracy. More importantly, in comparison with Cas9-mediated cell fitness 27 screens, BARBEKO screens are no longer affected by DNA-cleavage induced cytotoxicity in HeLa, 28 K562, or DSB-sensitive RPE1 cells. We anticipate that BARBEKO offers a valuable tool to 29 complement the current CRISPR screens in various settings