Author Correction: Genome-wide CRISPR screens identify ILF3 as a mediator of mTORC1-dependent amino acid sensing
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Autophagy repression by antigen and cytokines shapes mitochondrial, migration and effector machinery in CD8 T cells
Autophagy shapes CD8 T cell fate; yet the timing, triggers and targets of this process are poorly defined. Herein, we show that naive CD8 T cells have high autophagic flux, and we identify an autophagy checkpoint whereby antigen receptor engagement and inflammatory cytokines acutely repress autophagy by regulating amino acid transporter expression and intracellular amino acid delivery. Activated T cells with high levels of amino acid transporters have low autophagic flux in amino-acid-replete conditions but rapidly reinduce autophagy when amino acids are restricted. A census of proteins degraded and fueled by autophagy shows how autophagy shapes CD8 T cell proteomes. In cytotoxic T cells, dominant autophagy substrates include cytolytic effector molecules, and amino acid and glucose transporters. In naive T cells, mitophagy dominates and selective mitochondrial pruning supports the expression of molecules that coordinate T cell migration and survival. Autophagy thus differentially prunes naive and effector T cell proteomes and is dynamically repressed by antigen receptors and inflammatory cytokines to shape T cell differentiation.
A genome-wide atlas of human cell morphology
A key challenge of the modern genomics era is developing empirical data-driven representations of gene function. Here we present the first unbiased morphology-based genome-wide perturbation atlas in human cells, containing three genome-wide genotype–phenotype maps comprising CRISPR–Cas9-based knockouts of >20,000 genes in >30 million cells. Our optical pooled cell profiling platform (PERISCOPE) combines a destainable high-dimensional phenotyping panel (based on Cell Painting) with optical sequencing of molecular barcodes and a scalable open-source analysis pipeline to facilitate massively parallel screening of pooled perturbation libraries. This perturbation atlas comprises high-dimensional phenotypic profiles of individual cells with sufficient resolution to cluster thousands of human genes, reconstruct known pathways and protein–protein interaction networks, interrogate subcellular processes and identify culture media-specific responses. Using this atlas, we identify the poorly characterized disease-associated TMEM251/LYSET as a Golgi-resident transmembrane protein essential for mannose-6-phosphate-dependent trafficking of lysosomal enzymes. In sum, this perturbation atlas and screening platform represents a rich and accessible resource for connecting genes to cellular functions at scale.
CRISPR knock-in of a chimeric antigen receptor into GAPDH 3’UTR locus generates potent B7H3-specific NK-92MI cells
CAR-NK therapy is becoming a promising approach to treat solid tumors. However, the random insertion of the CAR gene and inflexible CAR expression caused by common preparation methods significantly impact its efficacy and safety. Here we successfully established a novel type of CAR-NK cells by integrating CAR sequences into the GAPDH 3’UTR locus of NK-92MI cells (CRISPR-CAR-NK), achieving site-specific integration of the CAR gene and allowing endogenous regulatory components to govern CAR expression. CRISPR-CAR-NK cells had comparable growth capacity but displayed superior anti-tumor activity compared with their lentiviral counterparts. They activated and degranulated more effectively when co-cultured with tumor cells, due to increased expression of activating receptors and decreased expression of inhibitory molecules. They also enhanced the production of Granzyme B and IFN-γ, and more effectively triggered the IFN-γ pathway. Moreover, CRISPR-CAR-NK cells demonstrated distinct properties from conventional CAR-NK concerning metabolic features and signal dependence. Notably, CRISPR-CAR-NK cells exhibited lower metabolic levels without compromising antitumor activity, and their function was less reliant on the PI3K-AKT pathway, implying that the CRISPR-CAR-NK cells have significant potential for enhanced synergy with AKT inhibitors and adaptation to nutrient stress within the tumor microenvironment. These findings provide a novel potential strategy for cancer immunotherapy and an experimental foundation and paradigm for optimizing CAR-NK cells utilizing CRISPR technology, highlighting the potential of CRISPR to advance immunotherapies.
The transcriptomic architecture of common cancers reflects synthetic lethal interactions
To maintain cell fitness, deleterious genetic alterations are buffered by compensatory changes in additional genes. In cancer, buffering processes could be targeted by synthetic lethality. However, despite the large-scale identification of synthetic lethal effects in preclinical models, evidence that these operate clinically is limited. This impedes the application of synthetic lethal approaches. By integrating molecular profiling data from >9,000 cancers with synthetic lethal screens, we show that transcriptomic buffering of tumor suppressor gene (TSG) loss by hyperexpression of synthetic lethal partners is a common phenomenon, extending to multiple TSGs and histotypes. Transcriptomic buffering is also notable in cancers that phenocopy TSG loss, such as BRCAness cancers, where expression of BRCA1/2 synthetic lethal genes correlates with clinical outcome. Synthetic lethal genes that exhibit transcriptomic buffering also represent more robust synthetic lethal effects. These observations have implications for understanding how tumor cells tolerate TSG loss, in part explain transcriptomic architectures in cancer and provide insight into target selection.
Amino acid auxotrophies in human gut bacteria are linked to higher microbiome diversity and long-term stability
Amino acid auxotrophies are prevalent among bacteria. They can govern ecological dynamics in microbial communities and indicate metabolic cross-feeding interactions among coexisting genotypes. Despite the ecological importance of auxotrophies, their distribution and impact on the diversity and function of the human gut microbiome remain poorly understood. This study performed the first systematic analysis of the distribution of amino acid auxotrophies in the human gut microbiome using a combined metabolomic, metagenomic, and metabolic modeling approach. Results showed that amino acid auxotrophies are ubiquitous in the colon microbiome, with tryptophan auxotrophy being the most common. Auxotrophy frequencies were higher for those amino acids that are also essential to the human host. Moreover, a higher overall abundance of auxotrophies was associated with greater microbiome diversity and stability, and the distribution of auxotrophs was found to be related to the human host’s metabolome, including trimethylamine oxide, small aromatic acids, and secondary bile acids. Thus, our results suggest that amino acid auxotrophies are important factors contributing to microbiome ecology and host-microbiome metabolic interactions.
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