Biology subjects

Worringer, K. A.

Publications and source records attributed to Worringer, K. A..

7 recordsLinked to original sources

bioRxiv2021

Patient brain organoids identify a link between the 16p11.2 copy number variant and the RBFOX1 gene.

Copy number variants (CNVs) that delete or duplicate 30 genes within the 16p11.2 genomic region give rise to a range of neurodevelopmental phenotypes with high penetrance in humans. Despite the identification of this small region, the mechanisms by which 16p11.2 CNVs lead to disease are unclear. Relevant models, like human cortical organoids (hCOs), are needed to understand the human-specific mechanisms of neurodevelopmental disease. We generated hCOs from 18 patients and controls, profiling 167,958 cells with single cell (sc)RNA-seq. Analysis revealed neuronal-specific differential expression of genes outside of the 16p11.2 region that were related to cell-cell adhesion, neuronal projection growth, and neurodevelopmental disorders. Furthermore, 16p11.2 deletion syndrome organoids exhibited reduced mRNA and protein levels of RBFOX1, a gene which can also harbor CNVs linked to neurodevelopmental phenotypes. We found that many genes previously shown to be regulated by RBFOX1 are also perturbed in organoids from patients with 16p11.2 deletion syndrome, and thus identified a novel link between independent CNVs associated with neuronal development and autism. Overall, this work suggests convergent signaling, which indicates the possibility of a common therapeutic mechanism across multiple rare neuronal diseases.

neuroscience↗

bioRxiv2021

Natural variation in gene expression and Zika virus susceptibility revealed by villages of neural progenitor cells

Variation in the human genome contributes to abundant diversity in human traits and vulnerabilities, but the underlying molecular and cellular mechanisms are not yet known, and will need scalable approaches to accelerate their recognition. Here, we advanced and applied an experimental platform that analyzes genetic, molecular, and phenotypic heterogeneity across cells from very many human donors cultured in a single, shared in vitro environment, with algorithms (Dropulation and Census-seq) for assigning phenotypes to individual donors. We used natural genetic variation and synthetic (CRISPR-Cas9) genetic perturbations to analyze the vulnerability of neural progenitor cells to infection with Zika virus. These analyses identified a common variant in the antiviral IFITM3 gene that regulated IFITM3 expression and explained most inter-individual variation in NPCs susceptibility to Zika virus infectivity. These and other approaches could provide scalable ways to recognize the impact of genes and genetic variation on cellular phenotypes. HIGHLIGHTSO_LIMeasuring cellular phenotypes in iPSCs and hPSC-derived NPCs from many donors C_LIO_LIEffects of donor sex, cell source, genetic and other variables on hPSC RNA expression C_LIO_LINatural genetic variation and synthetic perturbation screens both identify IFITM3 in NPC susceptibility to Zika virus C_LIO_LIA common genetic variant in IFITM3 explains most inter-individual variation in NPC susceptibility to Zika virus C_LI

genomics↗

bioRxiv2021

DRUG-seq Provides Unbiased Biological Activity Readouts for Drug Discovery

Unbiased transcriptomic RNA-seq data has provided deep insights about biological processes. However, its impact in drug discovery has been narrow given high costs and low throughput. Proof-of-concept studies with Digital RNA with pertUrbation of Genes (DRUG)-seq demonstrated the potential to address this gap. We extended the DRUG-seq platform by subjecting it to rigorous testing and by adding an open-source analysis pipeline. The results demonstrate high reproducibility and ability to resolve the mechanism(s) of action for a diverse set of compounds. Overall, the protocol and open-source analysis pipeline are a step towards industrializing RNA-seq for high complexity transcriptomics studies performed at a saturating scale.

genomics↗

bioRxiv2018

Genome-wide screens in accelerated human stem cell-derived neural progenitor cells identify Zika virus host factors and drivers of proliferation

Neural progenitor cells (NPCs) are essential to brain development and their dysfunction is linked to several disorders, including autism, Zika Virus Congenital Syndrome, and cancer. Understanding of these conditions has been improved by advancements with stem cell-derived NPC models. However, current differentiation methods require many days or weeks to generate NPCs and show variability in efficacy among cell lines. Here, we describe human Stem cell-derived NGN2-accelerated Progenitor cells (SNaPs), which are produced in only 48 hours. SNaPs express canonical forebrain NPC protein markers, are proliferative, multipotent, and like other human NPCs, are susceptible to Zika-mediated death. We further demonstrate SNaPs are valuable for large-scale investigations of genetic and environmental influencers of neurodevelopment by deploying them for genome-wide CRISPR-Cas9 screens. Our studies expand knowledge of NPCs by identifying known and novel Zika host factors, as well as new regulators of NPC proliferation validated by re-identification of the autism spectrum gene PTEN.

neuroscience↗

bioRxiv2018

Genome-Scale CRISPR Screening Identifies Novel Human Pluripotent Gene Networks

Human pluripotent stem cells (hPSCs) generate a wide variety of disease-relevant cells that can be used to improve the translation of preclinical research. Despite the potential of hPSCs, their use for genetic screening has been limited because of technical challenges. We developed a renewable Cas9/sgRNA-hPSC library where loss-of-function mutations can be induced at will. Our inducible-mutant hPSC library can be used for an unlimited number of genome-wide screens. We screened for novel genes involved in 3 of the fundamental properties of hPSCs: Their ability to self-renew/survive, their capacity to differentiate into somatic cells, and their inability to survive as single-cell clones. We identified a plethora of novel genes with unidentified roles in hPSCs. These results are available as a resource for the community to increase the understanding of both human development and genetics. In the future, our stem cell library approach will be a powerful tool to identify disease-modifying genes.\n\nVISUAL ABSTRACT\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC=\"FIGDIR/small/323436_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (48K):\norg.highwire.dtl.DTLVardef@5101e4org.highwire.dtl.DTLVardef@5321f2org.highwire.dtl.DTLVardef@1d9c94corg.highwire.dtl.DTLVardef@12ebd53_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2017

P53 toxicity is a hurdle to CRISPR/CAS9 screening and engineering in human pluripotent stem cells

CRISPR/Cas9 has revolutionized our ability to engineer genomes and to conduct genome-wide screens in human cells. While some cell types are easily modified with Cas9, human pluripotent stem cells (hPSCs) poorly tolerate Cas9 and are difficult to engineer. Using a stable Cas9 cell line or transient delivery of ribonucleoproteins (RNPs) we achieved an average insertion or deletion efficiency greater than 80%. This high efficiency made it apparent that double strand breaks (DSBs) induced by Cas9 are toxic and kill most treated hPSCs. Cas9 toxicity creates an obstacle to the high-throughput use CRISPR/Cas9 for genome-engineering and screening in hPSCs. We demonstrated the toxic response is tp53-dependent and the toxic effect of tp53 severely reduces the efficiency of precise genome-engineering in hPSCs. Our results highlight that CRISPR-based therapies derived from hPSCs should proceed with caution. Following engineering, it is critical to monitor for tp53 function, especially in hPSCs which spontaneously acquire tp53 mutations.

molecular biology↗

bioRxiv2017

ADAM17 Is The Main Sheddase For The Generation Of Human Triggering Receptor Expressed In Myeloid Cells (hTREM2) Ectodomain And Cleaves TREM2 After Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNF converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

cell biology↗