Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID-19), continues to cause substantial morbidity and mortality in the ongoing global pandemic. Understanding the fundamental mechanisms that govern innate immune and inflammatory responses during SARS-CoV-2 infection is critical for developing effective therapeutic strategies. Whereas interferon (IFN)–based therapies are generally expected to be beneficial during viral infection, clinical trials in COVID-19 have shown limited efficacy and potential detrimental effects of IFN treatment during SARS-CoV-2 infection. However, the underlying mechanisms responsible for this failure remain unknown. In this study, we found that IFN induced Z-DNA-binding protein 1 (ZBP1)–mediated inflammatory cell death, PANoptosis, in human and murine macrophages and in the lungs of mice infected with β-coronaviruses, including SARS-CoV-2 and mouse hepatitis virus (MHV). In patients with COVID-19, expression of the innate immune sensorZBP1was increased in immune cells from those who succumbed to the disease compared with those who recovered, further suggesting a link between ZBP1 and pathology. In mice, IFN-β treatment after β-coronavirus infection increased lethality, and genetic deletion ofZbp1or its Zα domain suppressed cell death and protected the mice from IFN-mediated lethality during β-coronavirus infection. Overall, our results identify that ZBP1 induced during coronavirus infection limits the efficacy of IFN therapy by driving inflammatory cell death and lethality. Therefore, inhibiting ZBP1 activity may improve the efficacy of IFN therapy, paving the way for the development of new and critically needed therapeutics for COVID-19 as well as other infections and inflammatory conditions where IFN-mediated cell death and pathology occur.

The T7 endonuclease 1 (T7E1) mismatch detection assay is a widely used method for evaluating the activity of site-specific nucleases, such as the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system. To determine the accuracy and sensitivity of this assay, we compared the editing estimates derived by the T7E1 assay with that of targeted next-generation sequencing (NGS) in pools of edited mammalian cells. Here, we report that estimates of nuclease activity determined by T7E1 most often do not accurately reflect the activity observed in edited cells. Editing efficiencies of CRISPR-Cas9 complexes with similar activity by T7E1 can prove dramatically different by NGS. Additionally, we compared editing efficiencies predicted by the Tracking of Indels by Decomposition (TIDE) assay and the Indel Detection by Amplicon Analysis (IDAA) assay to that observed by targeted NGS for both cellular pools and single-cell derived clones. We show that targeted NGS, TIDE, and IDAA assays predict similar editing efficiencies for pools of cells but that TIDE and IDAA can miscall alleles in edited clones.

DNMT3A deletion preserves CAR T cell functionality during prolonged stimulation.

CD8⁺ cytotoxic T cells (CTLs) orchestrate antitumour immunity and exhibit inherent heterogeneity^1,2, with precursor exhausted T (T_pex) cells but not terminally exhausted T (T_ex) cells capable of responding to existing immunotherapies^3–7. The gene regulatory network that underlies CTL differentiation and whether T_ex cell responses can be functionally reinvigorated are incompletely understood. Here we systematically mapped causal gene regulatory networks using single-cell CRISPR screens in vivo and discovered checkpoints for CTL differentiation. First, the exit from quiescence of T_pex cells initiated successive differentiation into intermediate T_ex cells. This process is differentially regulated by IKAROS and ETS1, the deficiencies of which dampened and increased mTORC1-associated metabolic activities, respectively. IKAROS-deficient cells accumulated as a metabolically quiescent T_pex cell population with limited differentiation potential following immune checkpoint blockade (ICB). Conversely, targeting ETS1 improved antitumour immunity and ICB efficacy by boosting differentiation of T_pex to intermediate T_ex cells and metabolic rewiring. Mechanistically, TCF-1 and BATF are the targets for IKAROS and ETS1, respectively. Second, the RBPJ–IRF1 axis promoted differentiation of intermediate T_ex to terminal T_ex cells. Accordingly, targeting RBPJ enhanced functional and epigenetic reprogramming of T_ex cells towards the proliferative state and improved therapeutic effects and ICB efficacy. Collectively, our study reveals that promoting the exit from quiescence of T_pex cells and enriching the proliferative T_ex cell state act as key modalities for antitumour effects and provides a systemic framework to integrate cell fate regulomes and reprogrammable functional determinants for cancer immunity.

Significance: Primary template-directed amplification (PTA) is a major improvement in whole genome amplification, which is required to study intratissue cellular evolution. As presented in the manuscript, PTA produces significantly improved and reproducible genome sequencing coverage and variant detection from a single genome of a single cell. Applications of measuring genetic diversity from single cells with PTA presented in the manuscript include examining the acquisition of genetic changes during normal development and aging, measuring the consequences of specific perturbations such as genome editing, and characterizing the evolution of clonal populations during cancer formation.