The prevalence of diabetes is increasing constantly, resulting in a global epidemic 1 . Diabetes is a major cause of blindness, kidney failure, heart attacks, stroke or lower limb amputation; in large parts because of marked changes in blood vessels, defined by expansion of the basement membrane and a loss of vascular cells [2][3][4] . Diabetes also impairs endothelial cell (EC) function 5 and disturbs EC-pericyte communication 6 . How endothelial/pericyte dysfunction leads to diabetic vasculopathy remains largely elusive. Here we report the development of self-organizing 3D human blood vessel organoids from pluripotent stem cells. These human blood vessel organoids contain endothelial cells and pericytes that self-assemble into capillary networks enveloped by a basement membrane. Human blood vessel organoids transplanted into mice form a stable, perfused vascular tree, including arteries, arterioles and venules. Exposure of blood vessel organoids to hyperglycemia and inflammatory cytokines in vitro induced thickening of the vascular basement membrane. Human blood vessels, exposed in vivo to a diabetic milieu in mice, also mimick the microvascular changes in diabetic patients. Dll4-Notch3 were identified as key

Loss-of-function (LOF) screens provide a powerful approach to identify regulators in biological processes. Pioneered in laboratory animals, LOF screens of human genes are currently restricted to two-dimensional (2D) cell culture hindering testing of gene functions requiring tissue context. Here we present CRISPR-LIneage tracing at Cellular resolution in Heterogenous Tissue (CRISPR-LICHT), enabling parallel LOF studies in human cerebral organoid tissue. We used CRISPR-LICHT to test 173 microcephaly candidate genes revealing 25 to be involved in known and uncharacterized microcephaly-associated pathways. We characterized Immediate Early Response 3 Interacting Protein 1 (IER3IP1) regulating the unfolded protein response (UPR) and extracellular matrix (ECM) protein secretion crucial for tissue integrity, with dysregulation resulting in microcephaly. Our human tissue screening technology identifies microcephaly genes and mechanisms involved in brain size control.

RNase P is the enzyme that removes 5′ extensions from tRNA precursors. With its diversity of enzyme forms—either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins—the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5′ or 3′ extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship.

The COVID-19 pandemic has demonstrated the need for massively-parallel, cost-effective tests monitoring viral spread. Here we present SARSeq, saliva analysis by RNA sequencing, a method to detect SARS-CoV-2 and other respiratory viruses on tens of thousands of samples in parallel. SARSeq relies on next generation sequencing of multiple amplicons generated in a multiplexed RT-PCR reaction. Two-dimensional, unique dual indexing, using four indices per sample, enables unambiguous and scalable assignment of reads to individual samples. We calibrate SARSeq on SARS-CoV-2 synthetic RNA, virions, and hundreds of human samples of various types. Robustness and sensitivity were virtually identical to quantitative RT-PCR. Double-blinded benchmarking to gold standard quantitative-RT-PCR performed by human diagnostics laboratories confirms this high sensitivity. SARSeq can be used to detect Influenza A and B viruses and human rhinovirus in parallel, and can be expanded for detection of other pathogens. Thus, SARSeq is ideally suited for differential diagnostic of infections during a pandemic.