BARBEKO

We use cytosine base editors for genome-scale knockout screens by targeting genes at their start codons, splice sites or 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 in making genome-scale CRISPR libraries (targeting 17,501 genes) in multiple human cell lines through lentiviral transduction at a high MOI (up to 10). With significantly reduced starting cells at high MOI, BARBEKO produces screening results with improved efficiency and accuracy. More importantly in comparison with Cas9-mediated genome-wide fitness screens, BARBEKO screens do not suffer from high copy number related confounding problems in both HeLa and K562 cells. Furthermore, we performed bidirectional fitness screens at high MOI in wild-type human retinal pigment epithelium-1 (RPE1) cells, which is a non-transformed cell line and sensitive to DSBs.