Since the project began two years ago, we have made significant progress toward all three of these goals.
1) We implemented a robust strategy for metabolic labeling of RNA using 4-TU and the SLAM-seq pipeline to differentiate old RNA from new RNA. This was a critical first step in generating temporal transcriptome trajectories. Additionally, our SLAM-seq analysis revealed that changes in both RNA maturation and stability are important, independent regulators of gene expression in T. brucei (Luzak et al, Nucleic Acids Research, 2025).
2) To generate high-resolution 3D maps of T. brucei genome architecture, we improved genome contiguity using ultra-long nanopore sequencing reads. We also implemented Micro-C, a high-resolution derivative of Hi-C, which revealed intriguing T. brucei organization around polymerase-class-specific transcriptional hubs. (Rabuffo et al. Nature Communications, 2024).
3) To improve our ability to conduct large-scale perturbation screens to identify regulators of cell-to-cell heterogeneity, we concentrated on three areas.
a) To obtain reliable transcriptome readouts, we developed a new, highly sensitive single-cell RNA-seq approach (Keneskhanova et al., Nature, 2025).
b) switching, we began implementing Cas9-based approaches for gene deletion and overexpression (unpublished).
c) To reliably measure VSG switching frequency at high resolution, we developed a strategy to grow T. brucei into micro populations on a large scale by encapsulating individual cells in semipermeable membranes (unpublished).
