Base-editing mutagenesis maps alleles to tune human T cell functions.

CRISPR-enabled screening is a powerful tool for the discovery of genes that control T cell function and has nominated candidate targets for immunotherapies1-6. However, new approaches are required to probe specific nucleotide sequences within key genes. Systematic mutagenesis in primary human T cells could reveal alleles that tune specific phenotypes. DNA base editors are powerful tools for introducing targeted mutations with high efficiency7,8. Here we develop a large-scale base-editing mutagenesis platform with the goal of pinpointing nucleotides that encode amino acid residues that tune primary human T cell activation responses. We generated a library of around 117,000 single guide RNA molecules targeting base editors to protein-coding sites across 385 genes implicated in T cell function and systematically identified protein domains and specific amino acid residues that regulate T cell activation and cytokine production. We found a broad spectrum of alleles with variants encoding critical residues in proteins including PIK3CD, VAV1, LCP2, PLCG1 and DGKZ, including both gain-of-function and loss-of-function mutations. We validated the functional effects of many alleles and further demonstrated that base-editing hits could positively and negatively tune T cell cytotoxic function. Finally, higher-resolution screening using a base editor with relaxed protospacer-adjacent motif requirements9 (NG versus NGG) revealed specific structural domains and protein-protein interaction sites that can be targeted to tune T cell functions. Base-editing screens in primary immune cells thus provide biochemical insights with the potential to accelerate immunotherapy design.

TMEM41B is a novel regulator of autophagy and lipid mobilization.

Autophagy maintains cellular homeostasis by targeting damaged organelles, pathogens, or misfolded protein aggregates for lysosomal degradation. The autophagic process is initiated by the formation of autophagosomes, which can selectively enclose cargo via autophagy cargo receptors. A machinery of well-characterized autophagy-related proteins orchestrates the biogenesis of autophagosomes; however, the origin of the required membranes is incompletely understood. Here, we have applied sensitized pooled CRISPR screens and identify the uncharacterized transmembrane protein TMEM41B as a novel regulator of autophagy. In the absence of TMEM41B, autophagosome biogenesis is stalled, LC3 accumulates at WIPI2- and DFCP1-positive isolation membranes, and lysosomal flux of autophagy cargo receptors and intracellular bacteria is impaired. In addition to defective autophagy, TMEM41B knockout cells display significantly enlarged lipid droplets and reduced mobilization and ß-oxidation of fatty acids. Immunostaining and interaction proteomics data suggest that TMEM41B localizes to the endoplasmic reticulum (ER). Taken together, we propose that TMEM41B is a novel ER-localized regulator of autophagosome biogenesis and lipid mobilization.

The pleiotropic deubiquitinase Ubp3 confers aneuploidy tolerance.

Aneuploidy-or an unbalanced karyotype in which whole chromosomes are gained or lost-causes reduced fitness at both the cellular and organismal levels but is also a hallmark of human cancers. Aneuploidy causes a variety of cellular stresses, including genomic instability, proteotoxic and oxidative stresses, and impaired protein trafficking. The deubiquitinase Ubp3, which was identified by a genome-wide screen for gene deletions that impair the fitness of aneuploid yeast, is a key regulator of aneuploid cell homeostasis. We show that deletion of UBP3 exacerbates both karyotype-specific phenotypes and global stresses of aneuploid cells, including oxidative and proteotoxic stress. Indeed, Ubp3 is essential for proper proteasome function in euploid cells, and deletion of this deubiquitinase leads to further proteasome-mediated proteotoxicity in aneuploid yeast. Notably, the importance of UBP3 in aneuploid cells is conserved. Depletion of the human homolog of UBP3, USP10, is detrimental to the fitness of human cells upon chromosome missegregation, and this fitness defect is accompanied by autophagy inhibition. We thus used a genome-wide screen in yeast to identify a guardian of aneuploid cell fitness conserved across species. We propose that interfering with Ubp3/USP10 function could be a productive avenue in the development of novel cancer therapeutics.

Chromosome-Specific and Global Effects of Aneuploidy in Saccharomyces cerevisiae.

Aneuploidy, an unbalanced karyotype in which one or more chromosomes are present in excess or reduced copy number, causes an array of known phenotypes including proteotoxicity, genomic instability, and slowed proliferation. However, the molecular consequences of aneuploidy are poorly understood and an unbiased investigation into aneuploid cell biology is lacking. We performed high-throughput screens for genes the deletion of which has a synthetic fitness cost in aneuploidy Saccharomyces cerevisiae cells containing single extra chromosomes. This analysis identified genes that, when deleted, decrease the fitness of specific disomic strains as well as those that impair the proliferation of a broad range of aneuploidies. In one case, a chromosome-specific synthetic growth defect could be explained fully by the specific duplication of a single gene on the aneuploid chromosome, highlighting the ability of individual dosage imbalances to cause chromosome-specific phenotypes in aneuploid cells. Deletion of other genes, particularly those involved in protein transport, however, confers synthetic sickness on a broad array of aneuploid strains. Indeed, aneuploid cells, regardless of karyotype, exhibit protein secretion and cell-wall integrity defects. Thus, we were able to use this screen to identify novel cellular consequences of aneuploidy, dependent on both specific chromosome imbalances and caused by many different aneuploid karyotypes. Interestingly, the vast majority of cancer cells are highly aneuploid, so this approach could be of further use in identifying both karyotype-specific and nonspecific stresses exhibited by cancer cells as potential targets for the development of novel cancer therapeutics.

Quantitative proteomic analysis reveals posttranslational responses to aneuploidy in yeast.

Aneuploidy causes severe developmental defects and is a near universal feature of tumor cells. Despite its profound effects, the cellular processes affected by aneuploidy are not well characterized. Here, we examined the consequences of aneuploidy on the proteome of aneuploid budding yeast strains. We show that although protein levels largely scale with gene copy number, subunits of multi-protein complexes are notable exceptions. Posttranslational mechanisms attenuate their expression when their encoding genes are in excess. Our proteomic analyses further revealed a novel aneuploidy-associated protein expression signature characteristic of altered metabolism and redox homeostasis. Indeed aneuploid cells harbor increased levels of reactive oxygen species (ROS). Interestingly, increased protein turnover attenuates ROS levels and this novel aneuploidy-associated signature and improves the fitness of most aneuploid strains. Our results show that aneuploidy causes alterations in metabolism and redox homeostasis. Cells respond to these alterations through both transcriptional and posttranscriptional mechanisms.

Aneuploidy causes proteotoxic stress in yeast.

Gains or losses of entire chromosomes lead to aneuploidy, a condition tolerated poorly in all eukaryotes analyzed to date. How aneuploidy affects organismal and cellular physiology is poorly understood. We found that aneuploid budding yeast cells are under proteotoxic stress. Aneuploid strains are prone to aggregation of endogenous proteins as well as of ectopically expressed hard-to-fold proteins such as those containing polyglutamine (polyQ) stretches. Protein aggregate formation in aneuploid yeast strains is likely due to limiting protein quality-control systems, since the proteasome and at least one chaperone family, Hsp90, are compromised in many aneuploid strains. The link between aneuploidy and the formation and persistence of protein aggregates could have important implications for diseases such as cancer and neurodegeneration.