Project description:Neuroblastoma cells were treated with a barcoded library of >600 genes and one of several standrard of care neuroblastoma compounds (or vehicle control)
Project description:Directed evolution in mammalian cells can facilitate the engineering of mammalian-compatible biomolecules and can enable synthetic evolvability for mammalian cells. We engineered an orthogonal alphaviral RNA replication system to evolve synthetic RNA-based devices, enabling RNA replicase-assisted continuous evolution (REPLACE) in live mammalian cells. To investigate the process of mutation accumulation in REPLACE system, we constructed a repRNA-v4 plasmid library containing 64 barcodes. Using this library, we analyzed the differences in mutation accumulation for different RNAs upon entry into cells, before and after molnupiravir treatment, and before and after FACS sorting. The results demonstrated that these barcoded RNAs undergo similar processes of mutation accumulation, providing evidence that mutations are commonly accumulated across different RNAs.
Project description:We describe "SBARRO" a system in which synaptic networks of cells are reconstructed from scRNA-seq data using clonal infectivity paths of barcoded rabies virus RNAs
Project description:Single cell genomics has revolutionized our understanding of the diversity of neuronal cell types. However, scalable technologies for probing single-cell connectivity are lacking, and we are just beginning to understand how molecularly defined cell types are organized into functional circuits. Here, we describe a strategy to generate high-complexity barcoded rabies virus (RV) for scalable circuit mapping that is compatible with both single-cell and single-nucleus RNA sequencing (sc/snRNA-seq) readout. Our barcoded RV libraries contain up to 43 million unique barcodes with a relatively uniform distribution, allowing multiplexed circuit mapping from tens of thousands of individual starter cells. We demonstrate the utility of this approach by mapping the emerging circuits in the developing human cortex using organotypic slice cultures. By leveraging the power and throughput of single cell genomics for mapping synaptic connectivity, we chart a path forward for scalable circuit mapping of molecularly-defined cell types in healthy and disease states.
Project description:Zika virus (ZIKV) is a mosquito-transmitted positive-sense RNA virus in the family Flaviviridae. ZIKV infections are associated with neurodevelopmental deficiencies termed Congenital Zika Syndrome. ZIKV strains are grouped into three phylogenetic lineages: East African, West African, and Asian, which contains the American lineage. RNA virus genomes exist as genetically-related sequences. The heterogeneity of these viral populations is implicated in viral fitness, and genome diversity is correlated to virulence. This study examines genetic diversity of representative ZIKV strains from all lineages utilizing next generation sequencing (NGS). Inter-lineage diversity results indicate that ZIKV lineages differ broadly from each other; however, intra-lineage comparisons of American ZIKV strains isolated from human serum or placenta show differences in diversity when compared to ZIKVs from Asia and West Africa. This study describes the first comprehensive NGS analysis of all ZIKV lineages and posits that sub-consensus-level diversity may provide a framework for understanding ZIKV fitness during infection.