CRISPR-based genome editing through the lens of DNA repair.

Genome editing technologies operate by inducing site-specific DNA perturbations that are resolved by cellular DNA repair pathways. Products of genome editors include DNA breaks generated by CRISPR-associated nucleases, base modifications induced by base editors, DNA flaps created by prime editors, and integration intermediates formed by site-specific recombinases and transposases associated with CRISPR systems. Here, we discuss the cellular processes that repair CRISPR-generated DNA lesions and describe strategies to obtain desirable genomic changes through modulation of DNA repair pathways. Advances in our understanding of the DNA repair circuitry, in conjunction with the rapid development of innovative genome editing technologies, promise to greatly enhance our ability to improve food production, combat environmental pollution, develop cell-based therapies, and cure genetic and infectious diseases.

Squalene accumulation in cholesterol auxotrophic lymphomas prevents oxidative cell death.

Cholesterol is essential for cells to grow and proliferate. Normal mammalian cells meet their need for cholesterol through its uptake or de novo synthesis1, but the extent to which cancer cells rely on each of these pathways remains poorly understood. Here, using a competitive proliferation assay on a pooled collection of DNA-barcoded cell lines, we identify a subset of cancer cells that is auxotrophic for cholesterol and thus highly dependent on its uptake. Through metabolic gene expression analysis, we pinpoint the loss of squalene monooxygenase expression as a cause of cholesterol auxotrophy, particularly in ALK+ anaplastic large cell lymphoma (ALCL) cell lines and primary tumours. Squalene monooxygenase catalyses the oxidation of squalene to 2,3-oxidosqualene in the cholesterol synthesis pathway and its loss results in accumulation of the upstream metabolite squalene, which is normally undetectable. In ALK+ ALCLs, squalene alters the cellular lipid profile and protects cancer cells from ferroptotic cell death, providing a growth advantage under conditions of oxidative stress and in tumour xenografts. Finally, a CRISPR-based genetic screen identified cholesterol uptake by the low-density lipoprotein receptor as essential for the growth of ALCL cells in culture and as patient-derived xenografts. This work reveals that the cholesterol auxotrophy of ALCLs is a targetable liability and, more broadly, that systematic approaches can be used to identify nutrient dependencies unique to individual cancer types.

MITO-Tag Mice enable rapid isolation and multimodal profiling of mitochondria from specific cell types in vivo.

Mitochondria are metabolic organelles that are essential for mammalian life, but the dynamics of mitochondrial metabolism within mammalian tissues in vivo remains incompletely understood. While whole-tissue metabolite profiling has been useful for studying metabolism in vivo, such an approach lacks resolution at the cellular and subcellular level. In vivo methods for interrogating organellar metabolites in specific cell types within mammalian tissues have been limited. To address this, we built on prior work in which we exploited a mitochondrially localized 3XHA epitope tag (MITO-Tag) for the fast isolation of mitochondria from cultured cells to generate MITO-Tag Mice. Affording spatiotemporal control over MITO-Tag expression, these transgenic animals enable the rapid, cell-type-specific immunoisolation of mitochondria from tissues, which we verified using a combination of proteomic and metabolomic approaches. Using MITO-Tag Mice and targeted and untargeted metabolite profiling, we identified changes during fasted and refed conditions in a diverse array of mitochondrial metabolites in hepatocytes and found metabolites that behaved differently at the mitochondrial versus whole-tissue level. MITO-Tag Mice should have utility for studying mitochondrial physiology, and our strategy should be generally applicable for studying other mammalian organelles in specific cell types in vivo.

One-pot DTECT enables rapid and efficient capture of genetic signatures for precision genome editing and clinical diagnostics.

The detection of genomic sequences and their alterations is crucial for basic research and clinical diagnostics. However, current methodologies are costly and time-consuming and require outsourcing sample preparation, processing, and analysis to genomic companies. Here, we establish One-pot DTECT, a platform that expedites the detection of genetic signatures, only requiring a short incubation of a PCR product in an optimized one-pot mixture. One-pot DTECT enables qualitative, quantitative, and visual detection of biologically relevant variants, such as cancer mutations, and nucleotide changes introduced by prime editing and base editing into cancer cells and human primary T cells. Notably, One-pot DTECT achieves quantification accuracy for targeted genetic signatures comparable with Sanger and next-generation sequencing. Furthermore, its effectiveness as a diagnostic platform is demonstrated by successfully detecting sickle cell variants in blood and saliva samples. Altogether, One-pot DTECT offers an efficient, versatile, adaptable, and cost-effective alternative to traditional methods for detecting genomic signatures.

Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia.

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.

A detection method for the capture of genomic signatures: From disease diagnosis to genome editing.

Variations in the genetic information originate from errors during DNA replication, error-prone repair of DNA damages, or genome editing. The most common approach to detect changes in DNA sequences employs sequencing technologies. However, they remain expensive and time-consuming, limiting their utility for routine laboratory experiments. We recently developed DinucleoTidE Signature CapTure (DTECT). DTECT is a marker-free and versatile detection method that captures targeted dinucleotide signatures resulting from the digestion of genomic amplicons by the type IIS restriction enzyme AcuI. Here, we describe the DTECT protocol to identify mutations introduced by CRISPR-based precision genome editing technologies or resulting from genetic variation. DTECT enables accurate detection of mutations using basic laboratory equipment and off-the-shelf reagents with qualitative or quantitative capture of signatures.

Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Among these, loss of heme synthesis reduces tumor growth due to a limiting role of heme in vivo, an effect independent of tissue origin or immune system. Our screens also identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. Altogether, this resource provides metabolic dependencies arising from microenvironmental limitations and the immune system, nominating potential anti-cancer targets.

Dietary thiamine influences l-asparaginase sensitivity in a subset of leukemia cells.

Tumor environment influences anticancer therapy response but which extracellular nutrients affect drug sensitivity is largely unknown. Using functional genomics, we determine modifiers of l-asparaginase (ASNase) response and identify thiamine pyrophosphate kinase 1 as a metabolic dependency under ASNase treatment. While thiamine is generally not limiting for cell proliferation, a DNA-barcode competition assay identifies leukemia cell lines that grow suboptimally under low thiamine and are characterized by low expression of solute carrier family 19 member 2 (SLC19A2), a thiamine transporter. SLC19A2 is necessary for optimal growth and ASNase resistance, when standard medium thiamine is lowered ~100-fold to human plasma concentrations. In addition, humanizing blood thiamine content of mice through diet sensitizes SLC19A2-low leukemia cells to ASNase in vivo. Together, our work reveals that thiamine utilization is a determinant of ASNase response for some cancer cells and that oversupplying vitamins may affect therapeutic response in leukemia.

ZBTB1 Regulates Asparagine Synthesis and Leukemia Cell Response to L-Asparaginase.

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response (ISR) that enables cell survival under nutrient stress. The mechanisms by which ATF4 couples metabolic stresses to specific transcriptional outputs remain unknown. Using functional genomics, we identified transcription factors that regulate the responses to distinct amino acid deprivation conditions. While ATF4 is universally required under amino acid starvation, our screens yielded a transcription factor, Zinc Finger and BTB domain-containing protein 1 (ZBTB1), as uniquely essential under asparagine deprivation. ZBTB1 knockout cells are unable to synthesize asparagine due to reduced expression of asparagine synthetase (ASNS), the enzyme responsible for asparagine synthesis. Mechanistically, ZBTB1 binds to the ASNS promoter and promotes ASNS transcription. Finally, loss of ZBTB1 sensitizes therapy-resistant T cell leukemia cells to L-asparaginase, a chemotherapeutic that depletes serum asparagine. Our work reveals a critical regulator of the nutrient stress response that may be of therapeutic value.

Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours.

As oxygen is essential for many metabolic pathways, tumour hypoxia may impair cancer cell proliferation1-4. However, the limiting metabolites for proliferation under hypoxia and in tumours are unknown. Here, we assessed proliferation of a collection of cancer cells following inhibition of the mitochondrial electron transport chain (ETC), a major metabolic pathway requiring molecular oxygen5. Sensitivity to ETC inhibition varied across cell lines, and subsequent metabolomic analysis uncovered aspartate availability as a major determinant of sensitivity. Cell lines least sensitive to ETC inhibition maintain aspartate levels by importing it through an aspartate/glutamate transporter, SLC1A3. Genetic or pharmacologic modulation of SLC1A3 activity markedly altered cancer cell sensitivity to ETC inhibitors. Interestingly, aspartate levels also decrease under low oxygen, and increasing aspartate import by SLC1A3 provides a competitive advantage to cancer cells at low oxygen levels and in tumour xenografts. Finally, aspartate levels in primary human tumours negatively correlate with the expression of hypoxia markers, suggesting that tumour hypoxia is sufficient to inhibit ETC and, consequently, aspartate synthesis in vivo. Therefore, aspartate may be a limiting metabolite for tumour growth, and aspartate availability could be targeted for cancer therapy.

Publisher Correction: Aspartate is a limiting metabolite for cancer cell proliferation under hypoxia and in tumours.

In the version of this Letter originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Letter.