A5064525281- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- Pluripotent Stem Cells Research
- RNA and protein synthesis mechanisms
- Genetics, Aging, and Longevity in Model Organisms
- Protein Tyrosine Phosphatases
- Wnt/β-catenin signaling in development and cancer
- Autophagy in Disease and Therapy
- Coagulation, Bradykinin, Polyphosphates, and Angioedema
- Innovation and Socioeconomic Development
- Renal and related cancers
- Single-cell and spatial transcriptomics
- Synthesis of heterocyclic compounds
- Synthesis and Reactivity of Sulfur-Containing Compounds
Institute of Molecular Biotechnology
2023-2024
Vienna Biocenter
2024
Medical University of Vienna
2024
ToolGen (South Korea)
2017-2023
Korea Advanced Institute of Science and Technology
2021
Abstract The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated often poorly efficient in human cells. Here, we develop directed evolution approach E. coli to obtain Sniper-Cas9, which shows high without killing on-target activities Unlike other engineered variants, Sniper-Cas9 WT-level with...
Abstract Although several high-fidelity SpCas9 variants have been reported, it has observed that this increased specificity is associated with reduced on-target activity, limiting the applications of when efficient genome editing required. Here, we developed an improved version Sniper–Cas9, Sniper2L, which represents exception to trade-off trend as showed higher retained high activity. We evaluated Sniper2L activities at a large number target sequences and DeepSniper, deep learning model can...
The pluripotency of embryonic stem cells (ESCs) is maintained by intracellular networks many pluripotency-associated (PA) proteins such as OCT4, SOX2, and NANOG. However, the mechanisms underlying regulation protein homeostasis for remain elusive. Here, we first demonstrate that autophagy acts together with ubiquitin-proteasome system (UPS) to modulate levels PA in human ESCs (hESCs). Autophagy inhibition impaired despite increment hESCs. Immunogold-electron microscopy confirmed localization...
Ascl1 and Ngn2, closely related proneural transcription factors, are able to convert mouse embryonic stem cells into induced neurons. Despite their similarities, these factors elicit only partially overlapping transcriptional programs, it remains unknown whether converted via distinct mechanisms. Here we show that Ngn2 induce mutually exclusive side populations by binding activating lineage drivers. Furthermore, rapidly dismantles the pluripotency network installs neuronal trophoblast cell...
Pooled genetic screening with CRISPR–Cas9 has enabled genome-wide, high-resolution mapping of genes to phenotypes, but assessing the effect a given perturbation requires evaluation each single guide RNA (sgRNA) in hundreds cells counter stochastic drift and obtain robust results. However, resolution is limited complex, heterogeneous models, such as organoids or tumors transplanted into mice, because achieving sufficient representation impractical scaling. This due bottleneck effects...
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance its potentially deleterious off-target effects. Several methods have been devised to reduce such Here, we present an Escherichia coli-based directed evolution method called Sniper-screen obtain a Cas9 variant with optimized specificity and retained on-target activity, Sniper-Cas9. Using Sniper-screen, positive negative selection...
Costello syndrome (CS) is an autosomal dominant disorder caused by mutations in HRAS. Although CS patients have skeletal abnormalities, the role of mutated HRAS bone development remains unclear. Here, we use induced pluripotent stem cells (iPSCs) undergoing osteogenic differentiation to investigate how dysregulation extracellular matrix (ECM) remodeling proteins contributes impaired osteogenesis. patient-derived iPSCs develop normally produce mesenchymal (MSCs), resulting MSCs show defective...
Abstract The use of CRISPR-Cas9 as a therapeutic reagent is hampered by its off-target effects. Although rationally designed S. pyogenes Cas9 (SpCas9) variants that display higher specificities than the wild-type SpCas9 protein are available, these attenuated often poorly efficient in human cells. Here, we have used directed evolution approach E. coli to obtain Sniper-Cas9, which shows high without sacrificing on-target activities
The development of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (Cas9) into therapeutic modalities requires the avoidance its potentially deleterious off-target effects. Several methods have been devised to reduce such Here, we present an Escherichia coli-based directed evolution method called Sniper-screen obtain a Cas9 variant with optimized specificity and retained on-target activity, Sniper-Cas9. Using Sniper-screen, positive negative selection...
Abstract Although several high-fidelity SpCas9 variants that have reduced activities at mismatched target sequences been reported, it has observed this increased specificity is associated with on-target activity, limiting the applications of when efficient genome editing required. Here, we developed an improved version Sniper-Cas9, Sniper2L, which represents exception to trade-off trend as showed higher retained high activity. We evaluated Sniper2L a large number sequences, and DeepSniper,...