Spatiotemporal analysis of human intestinal development at single-cell resolution.

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work.

Tracking in situ checkpoint inhibitor-bound target T cells in patients with checkpoint-induced colitis.

The success of checkpoint inhibitors (CPIs) for cancer has been tempered by immune-related adverse effects including colitis. CPI-induced colitis is hallmarked by expansion of resident mucosal IFNγ cytotoxic CD8+ T cells, but how these arise is unclear. Here, we track CPI-bound T cells in intestinal tissue using multimodal single-cell and subcellular spatial transcriptomics (ST). Target occupancy was increased in inflamed tissue, with drug-bound T cells located in distinct microdomains distinguished by specific intercellular signaling and transcriptional gradients. CPI-bound cells were largely CD4+ T cells, including enrichment in CPI-bound peripheral helper, follicular helper, and regulatory T cells. IFNγ CD8+ T cells emerged from both tissue-resident memory (TRM) and peripheral populations, displayed more restricted target occupancy profiles, and co-localized with damaged epithelial microdomains lacking effective regulatory cues. Our multimodal analysis identifies causal pathways and constitutes a resource to inform novel preventive strategies.

Isolation of human fetal intestinal cells for single-cell RNA sequencing.

The intestine has a large number of cell types. Thus, digestion of pure and viable populations is necessary for downstream techniques including single-cell RNA sequencing. We outline a protocol to isolate both epithelial and non-epithelial cells from human fetal samples at high viability, which was used to produce a full thickness atlas of intestinal cells across human development. This protocol can also be adapted to adult endoscopy and surgical specimens. For details on the use of this protocol, please refer to Fawkner-Corbett et al. (2021).

Keeping crispr in check: diverse mechanisms of phage-encoded anti-crisprs.

CRISPR-Cas represents the only adaptive immune system of prokaryotes known to date. These immune systems are widespread among bacteria and archaea, and provide protection against invasion of mobile genetic elements, such as bacteriophages and plasmids. As a result of the arms-race between phages and their prokaryotic hosts, phages have evolved inhibitors known as anti-CRISPR (Acr) proteins to evade CRISPR immunity. In the recent years, several Acr proteins have been described in both temperate and virulent phages targeting diverse CRISPR-Cas systems. Here, we describe the strategies of Acr discovery and the multiple molecular mechanisms by which these proteins operate to inhibit CRISPR immunity. We discuss the biological relevance of Acr proteins and speculate on the implications of their activity for the development of improved CRISPR-based research and biotechnological tools.