A5022832714- CRISPR and Genetic Engineering
- Genomics and Chromatin Dynamics
- Protein Degradation and Inhibitors
- Epigenetics and DNA Methylation
- Viral Infectious Diseases and Gene Expression in Insects
- RNA and protein synthesis mechanisms
- Lung Cancer Treatments and Mutations
- Cancer Genomics and Diagnostics
- Gene Regulatory Network Analysis
- Cell Image Analysis Techniques
- Neuroscience and Neuropharmacology Research
- Virus-based gene therapy research
- Amyotrophic Lateral Sclerosis Research
- RNA Interference and Gene Delivery
- Ubiquitin and proteasome pathways
- Single-cell and spatial transcriptomics
- RNA modifications and cancer
- RNA Research and Splicing
- Mitochondrial Function and Pathology
- Cellular transport and secretion
- Click Chemistry and Applications
- Inflammation biomarkers and pathways
- 3D Printing in Biomedical Research
- Lysosomal Storage Disorders Research
- Parkinson's Disease Mechanisms and Treatments
Stanford University
2019-2024
Bioengineering Center
2021-2022
University of California, San Francisco
2017-2020
Chan Zuckerberg Initiative (United States)
2019
Gladstone Institutes
2017-2018
Highlights•A CRISPR interference platform for genetic screens in human iPSC-derived neurons•Survival uncover genes essential neurons, but not iPSCs or cancer cells•Single-cell RNA-seq reveal distinct neuronal roles ubiquitous genes•Arrayed high-content controlling morphologySummaryCRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based were conducted lines rather than healthy, differentiated cells. Here, we...
Regulatory T cells (Tregs), which are characterized by expression of the transcription factor Foxp3, a dynamic and heterogeneous population that control immune responses prevent autoimmunity. We recently identified subset Tregs in murine skin with properties typical memory defined this as (mTregs). Due to importance these regulating tissue inflammation mice, we analyzed cell humans found almost all normal had an activated phenotype. Compared mTregs peripheral blood, cutaneous unique surface...
Lowering total tau levels is an attractive therapeutic strategy for Alzheimer's disease and other tauopathies. High-throughput screening in neurons derived from human induced pluripotent stem cells (iPSCs) a powerful tool to identify tau-targeted therapeutics. However, such screens have been hampered by heterogeneous neuronal production, high cost low yield, multi-step differentiation procedures. We engineered isogenic iPSC line that harbors inducible neurogenin 2 transgene, transcription...
How dipeptide repeats cause pathology A repeat expansion in the chromosome 9 open reading frame 72 ( C9orf72 ) gene is most common known of two neurodegenerative diseases: frontotemporal dementia and amyotrophic lateral sclerosis. This leads to abnormal production proteins repeating dipeptides, but their contribution disease pathogenesis remains unclear. Zhang et al. engineered a mouse model study consequences one these dipeptides—prolinearginine protein, poly(PR)—in brain. They found that...
Heterozygous mutations in the GRN gene lead to progranulin (PGRN) haploinsufficiency and cause frontotemporal dementia (FTD), a neurodegenerative syndrome of older adults. Homozygous mutations, on other hand, complete PGRN loss neuronal ceroid lipofuscinosis (NCL), lysosomal storage disease usually seen children. Given that predominant clinical pathological features FTD NCL are distinct, it is controversial whether mechanisms associated with partial similar or distinct. We show leads...
Alzheimer’s disease (AD), the most common form of dementia, is characterized by abnormal accumulation amyloid plaques and hyperphosphorylated tau aggregates, as well microgliosis. Hemizygous missense variants in Triggering Receptor Expressed on Myeloid Cells 2 ( TREM2 ) are associated with elevated risk for developing late-onset AD. These hypothesized to result loss function, mimicking haploinsufficiency. However, consequences haploinsufficiency pathology microglial function remain unknown....
Human astrocytes network with neurons in dynamic ways that are still poorly defined. Our ability to model this relationship is hampered by the lack of relevant and convenient tools recapitulate complex interaction. To address barrier, we have devised efficient coculture systems utilizing 3D organoid-like spheres, termed asteroids, containing pre-differentiated human pluripotent stem cell (hPSC)-derived (hAstros) combined generated from hPSC-derived neural cells (hNeurons) or directly induced...
Abstract Human nuclear proteins contain >1000 transcriptional effector domains that can activate or repress transcription of target genes. We lack a systematic understanding which regulate robustly across genomic, cell-type, and DNA-binding domain (DBD) contexts. Here, we developed dCas9-mediated high-throughput recruitment (HT-recruit), pooled screening method for quantifying function at endogenous targets, tested library containing 5092 protein Pfam varied find many effectors depend on...
In mammalian cells genes that are in close proximity can be transcriptionally coupled: silencing or activating one gene affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, when designing synthetic regulation circuits. Here, we systematically dissect this process single by recruiting releasing repressive chromatin regulators at dual-gene reporters, measuring how fast reactivation spread a...
Abstract Rhodopsin ( RHO ) missense variants are a leading cause of autosomal dominant retinitis pigmentosa (adRP), progressive retinal degeneration with no currently approved therapies. Interpreting the pathogenicity growing number identified is major clinical challenge, and understanding their disease mechanisms essential for developing effective Here, we present high-resolution map variant trafficking using two complementary deep mutational scanning (DMS) approaches based on surface...
Summary Human gene expression is regulated by over two thousand transcription factors and chromatin regulators 1,2 . Effector domains within these proteins can activate or repress transcription. However, for many of we do not know what type transcriptional effector they contain, their location in the protein, activation repression strengths, amino acids that are necessary functions. Here, systematically measure activity >100,000 protein fragments (each 80 long) tiling across most human...
Histone H3.3 is frequently mutated in cancers, with the lysine 36 to methionine mutation (K36M) being a hallmark of chondroblastomas. While it known that H3.3K36M changes cellular epigenetic landscape, remains unclear how affects dynamics gene expression. Here, we use synthetic reporter measure effect on silencing and memory after recruitment KRAB: member largest class human repressors, commonly used biology, associated H3K9me3. We find H3.3K36M, which decreases H3K36 methylation, leads...
SUMMARY CRISPR/Cas9-based functional genomics have transformed our ability to elucidate mammalian cell biology. However, most previous CRISPR-based screens were conducted in cancer lines, rather than healthy, differentiated cells. Here, we describe a CRISPR interference (CRISPRi)-based platform for genetic human neurons derived from induced pluripotent stem cells (iPSCs). We demonstrate robust and durable knockdown of endogenous genes such neurons, present results three complementary...
Summary Viruses encode transcriptional regulatory proteins critical for controlling viral and host gene expression. Given their multifunctional nature high sequence divergence, it is unclear which can affect transcription specific sequences contribute to this function. Using a high-throughput assay, we measured the potential of over 60,000 protein tiles across ∼1,500 from 11 coronaviruses all nine human herpesviruses. We discovered hundreds new effector domains, including conserved...
Abstract Controlling gene expression and chromatin state via the recruitment of transcriptional effector proteins to specific genetic loci has advanced potential mammalian synthetic biology, but is still hindered by challenge delivering large regulators. Here, we develop a new method for generating small nanobodies against human regulators that can repress or activate expression. We start with diverse nanobody library perform enrichment regulatory complexes using yeast display, followed...
Abstract Despite growing knowledge of the functions individual human transcriptional effector domains, much less is understood about how multiple domains within same protein combine to regulate gene expression. Here, we measure activity for 8,400 domain combinations by recruiting them reporter genes in cells. In our assay, weak and moderate activation synergize drive strong expression, while combining activators often results weaker activation. contrast, repressors linearly produce full...
This protocol describes thedifferentiation of iPSCs with stably integrated doxycycline-inducible Ngn2 (such as i3Ns).
Abstract In mammalian cells genes that are in close proximity coupled transcriptionally: silencing or activating one gene can affect its neighbors. Understanding these dynamics is important for natural processes, such as heterochromatin spreading during development and aging, when designing synthetic regulation. Here, we systematically dissect this process single by recruiting releasing repressive chromatin regulators at dual-gene reporters, measuring how fast reactivation spread a function...
This protocol explains general culture and maintenance of the WTc11 iPSC cell line.