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Abstract Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screening platform by generating a DNA damage response (DDR)-focused custom sgRNA library targeting 1011 genes with 3033 sgRNAs. We performed five proof-of-principle compound screens and found that the compounds’ known modes-of-action (MoA) were enriched among the compounds’ CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale with 20-fold fewer resources than commonly used genome-wide libraries and produce biologically informative CGI profiles.

Abstract Most eukaryotic proteins are N-terminally acetylated, but the functional impact on a global scale has remained obscure. Using genome-wide CRISPR knockout screens in human cells, we reveal a strong genetic dependency between a major N-terminal acetyltransferase and specific ubiquitin ligases. Biochemical analyses uncover that both the ubiquitin ligase complex UBR4-KCMF1 and the acetyltransferase NatC recognize proteins bearing an unacetylated N-terminal methionine followed by a hydrophobic residue. NatC KO-induced protein degradation and phenotypes are reversed by UBR knockdown, demonstrating the central cellular role of this interplay. We reveal that loss of Drosophila NatC is associated with male sterility, reduced longevity, and age-dependent loss of motility due to developmental muscle defects. Remarkably, muscle-specific overexpression of UbcE2M, one of the proteins targeted for NatC KO-mediated degradation, suppresses defects of NatC deletion. In conclusion, NatC-mediated N-terminal acetylation acts as a protective mechanism against protein degradation, which is relevant for increased longevity and motility.

Here, the authors perform a large-scale, high-throughput biochemical assay to determine the compatibility of over 300,000 domain recombination variants of the inward rectifier K+ channel Kir2.1. They derive rules for designing domain insertion variants that fold and traffic to the cell surface and conclude that the insertion of domains at protein termini is evolutionary favoured.

The analysis of essential genes in pathogens can be used to discover potential antimicrobial targets. Here, the authors use a machine learning model and chemogenomic analyses to generate genome-wide gene essentiality predictions for the fungal pathogen Candida albicans, define the function of three uncharacterized essential genes, and identify the target of a new antifungal compound.

Genetic interactions may contribute to phenotypic traits but are challenging to decipher. Here, the authors develop BridGE, a computational approach for identifying pathways connected by genetic interactions from GWAS data.

Allostery is a fundamental principle of protein regulation that remains challenging to engineer. Here authors screen human Inward Rectifier K + Channel Kir2.1 for permissibility to domain insertions and propose that differential permissibility is a metric of latent allosteric capacity in Kir2.1.

The mechanisms of action of the antibacterial metalloid tellurite are unclear. Here, the authors show that tellurite induces an accumulation of hydroxyl radical and intermediates of heme biosynthesis inE. coli, and that the heme precursor 5-aminolevulinic acid potentiates tellurite toxicity.