Abstract
The yeast knockout library, comprised of strains carrying precise deletions of individual open reading frames (ORFs), has been a vital resource to understand gene function. However, accumulation of suppressor mutations in knockout strains can lead to erroneous functional annotations. Moreover, the library is limited to non-essential ORFs, and must be complemented with hypomorphic alleles of essential ORFs for genome- wide studies. To address these limitations, we constructed genome-wide libraries of conditional alleles based on the auxin-inducible degron (AID) system for conditional degradation of AID-tagged proteins. First, we determined that N-terminal tagging is at least twice more likely to inadvertently impair protein function across the proteome. We thus constructed two genome-wide libraries with over 5600 essential and non-essential proteins fused at the C-terminus with an AID tag and an optional fluorescent protein. Almost 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 with the AID libraries revealed a role for the glucose signaling factor GSF2 in resistance to hydroxyurea, highlighting how these resources extend the toolbox for functional genomics in yeast.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Major points Quantification of the fitness impact of the GAL1-OsTIR1(F74G) construct and correction of the strain fitness data of the AID-v1 library for this effect. Estimation of the detection limit of the colony-based fluorescence measurements. Revised section on screens for DNA damage response factors, to clarify the number of essential hits and show their validation. Demonstration that the gsf2∆ mutant exhibits delayed cell cycle progression after release from hydroxyurea. Clarifications of experimental design and discussion.
