Ontology highlight
ABSTRACT:
PROVIDER: PRJNA1302297 | ENA |
REPOSITORIES: ENA
Similar Datasets
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. Toinvestigatetheexpressionheterogeneityofself-replicatingRNAsinrepRNA-v4cells,weperformedsingle-cellRNA-seqanalysisusingthe10xGenomicssequencingmethod.Ouranalysisofthesingle-cellRNA-seqprofilingdatarevealedarelativelyuniformexpressionpatternofself-replicatingRNAswithinrepRNA-v4cells.
2024-07-07 | GSE235341 | GEO
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. Using REPLACE, we attempted continuous intracellular evolution of the negative dominant mutant KRAS (S17N). To analyze the process of mutation accumulation, we performed amplicon sequencing on experimental materials at different stages and under different treatment conditions. The results indicated that the mutations generated by this system were primarily induced by Monanunavir, and the addition of Monanunavir significantly accelerated the rate of evolution.
2024-07-07 | GSE235327 | GEO
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. we employed REPLACE to drive the continuous intracellular evolution of the cancer-related protein MEK1 with the aim of conferring resistance to Cobimetinib. To investigate the accumulation of mutations during this evolutionary process, we conducted amplicon sequencing on experimental materials collected at different stages. The results revealed intricate relationships among different mutations, highlighting the complex nature of the evolutionary landscape.
2024-07-07 | GSE235328 | GEO
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. RNA-seq of 3 different cells was performed to analyze the effect of introducing different versions of RNA replication systems (i.e., repRNA-v3 and repRNA-v4) into BHK-21 cells on endogenous gene expression. Analysis of the transcriptome profiling data indicated that repRNA-v4 stimulated lower interferon signaling and may have lower cytopathicity.
2024-07-07 | GSE217397 | GEO
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 determine the degradation rate of alphaviral RNA in BHK-21 cells, 10 µg of repRNA-v4 RNA was transfected into 4 million wildtype BHK-21 cells via electroporation. Samples were collected at 1-hour, 3-hour, and 12-hour time points for subsequent RNA-seq analysis. The findings revealed that the half-life of alphaviral RNA in the cells was approximately 3.5 hours.
2024-07-07 | GSE235339 | GEO
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.
2024-07-07 | GSE235338 | GEO
Project description:Cell surface proteins (CSP) are integral to cellular function and intercellular communication in complex tissues and represent over 70% of drug targets. Yet, no robust method exists for mapping CSPs in living organisms. While proximity labeling techniques have advanced our understanding of molecular interactions across the cell, our ability to tag the extracellular proteome of specific cell types in vivo remains lacking. Here, we present TORCH (Tyrosinase Oxidizer for Rapid Chemical Highlighting), an engineered tyrosinase enzyme enabling rapid, genetically targeted proximity labeling of cell surface proteomes in living organisms, using only ambient oxygen as a cofactor. Developed through directed evolution and structure-guided engineering, TORCH achieves ~15-fold increased efficiency and robust mammalian expression compared to wild-type tyrosinase. Beyond cell-intrinsic profiling, TORCH's rapid kinetics enable detection of transient cell-cell interactions by labeling proteins at contact interfaces. We demonstrate TORCH’s utility by mapping immune cell interactions in tumor microenvironments and profiling blood-brain barrier (BBB) endothelial cell surface proteomes directly in living mice. BBB profiling uncovered PRNP as a novel GPI-anchored transport receptor, opening new avenues for CNS drug delivery. Overall, TORCH provides a robust and versatile platform for decoding cell-type-specific surface proteomes and cell-cell interactions within native physiological contexts.
2026-09-23 | PXD078371 | Pride
Project description:Cell surface proteins (CSP) are integral to cellular function and intercellular communication in complex tissues and represent over 70% of drug targets. Yet, no robust method exists for mapping CSPs in living organisms. While proximity labeling techniques have advanced our understanding of molecular interactions across the cell, our ability to tag the extracellular proteome of specific cell types in vivo remains lacking. Here, we present TORCH (Tyrosinase Oxidizer for Rapid Chemical Highlighting), an engineered tyrosinase enzyme enabling rapid, genetically targeted proximity labeling of cell surface proteomes in living organisms, using only ambient oxygen as a cofactor. Developed through directed evolution and structure-guided engineering, TORCH achieves ~15-fold increased efficiency and robust mammalian expression compared to wild-type tyrosinase. Beyond cell-intrinsic profiling, TORCH's rapid kinetics enable detection of transient cell-cell interactions by labeling proteins at contact interfaces. We demonstrate TORCH’s utility by mapping immune cell interactions in tumor microenvironments and profiling blood-brain barrier (BBB) endothelial cell surface proteomes directly in living mice. BBB profiling uncovered PRNP as a novel GPI-anchored transport receptor, opening new avenues for CNS drug delivery. Overall, TORCH provides a robust and versatile platform for decoding cell-type-specific surface proteomes and cell-cell interactions within native physiological contexts.
2026-09-23 | PXD068212 | Pride