Functional genomics with libraries of knockout alleles is limited to non-essential genes and convoluted by the potential accumulation of suppressor mutations in knockout backgrounds, which can lead to erroneous functional annotations. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for inducible degradation of AID-tagged proteins in the budding yeast Saccharomyces cerevisiae. First, we determined that N-terminal tagging is at least twice as likely to inadvertently impair protein function across the proteome. We thus constructed two libraries with over 5,600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Approximately 90% of AID-tagged proteins were degraded in the presence of the auxin analog 5-Ph-IAA, with initial protein abundance and tag accessibility as limiting factors. Genome-wide screens for DNA damage response factors revealed a role for the glucose signaling factor GSF2 in resistance to hydroxyurea, highlighting how the AID libraries extend the yeast genetics toolbox.
© 2024 Gameiro et al.