Fig. 2. A gRNA library for systematic perturbation of essential genes using the Target-AID base editor.

Essential genes (ex.: E.G.1) were scanned for sites appropriate for Target-AID mutagenesis. Mutational outcomes include silent (gray triangle), missense (black triangle) mutations, as well as stop codons (*). DNA fragments corresponding to the gRNA sequences were synthesized as an oligonucleotide pool and cloned into a co-selection base editing vector. Using gRNAs as molecular barcodes, the abundance of cell subpopulations bearing mutations is then measured after mutagenesis and bulk competition. Mutations with fitness effects are inferred from reductions in the relative gRNA abundances.