Lingting Shi

In vivo single-cell CRISPR screens can resolve perturbation effects in native tissues, but few combine cell-type-restricted delivery, somatic tumor initiation, and combinatorial perturbation. Here we developed an RCAS-TVA-based single-cell CRISPR screening approach coupling oncogenic transformation with single- or dual-guide perturbation in defined neural progenitors. In glioma, tumor suppressor loss produced distinct genotype-dependent transcriptional states, including an NF1-loss-associated increase in mesenchymal programs. Introducing a fixed Cdkn2a(ARF) perturbation altered malignant-cell state composition, immune-cell abundance, and the effects of additional tumor-suppressor perturbations, including increased representation of Pten- and Rb1-deficient cells, suggestive of the relief of an ARF-dependent genetic barrier. These perturbations converged on transcriptional states resembling pharmacologic p53 activation in human glioblastoma. Across mouse tumors and human glioblastoma models, this response was associated with increased astrocyte-like state representation. These findings establish a framework for single-cell analysis of context-dependent somatic perturbations during tumor initiation in vivo.

Single-cell RNA sequencing has transformed the field of cellular biology by providing unprecedented insights into cellular heterogeneity. However, characterizing scRNA-seq datasets remains a significant challenge. We introduce RefCM, a novel computational method that combines optimal transport and integer programming to enhance the annotation of scRNA clusters using established reference datasets. Our method produces highly accurate cross-technology, cross-tissue, and cross-species mappings while remaining tractable at atlas scale, outperforming existing methods across all these tasks. By providing precise annotations, RefCM can enable the discovery of new cell types, states, and relationships in single-cell transcriptomic data.

Induced Tregs (iTregs) have great promise in adoptive immunotherapy for treatment of autoimmune diseases. This report investigates the impacts of substrate stiffness on human Treg induction, providing a powerful yet simple approach to improving production of these cells. Conventional CD4⁺ human T cells were activated on materials of different elastic modulus and cultured under suppressive conditions. Enhanced Treg induction was observed on softer materials as early as 3 days following activation and persisted for multiple weeks. Substrate stiffness also affected epigenetic modification of Treg specific genes and Treg suppressive capacity. Tregs induced on substrates of an optimal stiffness balance quantity and suppressive quality.