A5090442852- RNA and protein synthesis mechanisms
- Endoplasmic Reticulum Stress and Disease
- CRISPR and Genetic Engineering
- Mitochondrial Function and Pathology
- Advanced biosensing and bioanalysis techniques
- Cellular transport and secretion
- Protein Structure and Dynamics
- RNA modifications and cancer
- RNA Interference and Gene Delivery
- Congenital heart defects research
- Photoreceptor and optogenetics research
- Pancreatic function and diabetes
- Ubiquitin and proteasome pathways
- Circadian rhythm and melatonin
- Lipid Membrane Structure and Behavior
- RNA regulation and disease
- RNA Research and Splicing
- Genetics and Neurodevelopmental Disorders
- Ion Transport and Channel Regulation
- Genomic variations and chromosomal abnormalities
- Single-cell and spatial transcriptomics
- Chromosomal and Genetic Variations
- Genetics, Aging, and Longevity in Model Organisms
- Bacterial Genetics and Biotechnology
- interferon and immune responses
California Institute of Technology
2019-2024
Whitehead Institute for Biomedical Research
2021-2024
Massachusetts Institute of Technology
2021-2023
University of California, San Francisco
2021-2022
City College of San Francisco
2020
MRC Laboratory of Molecular Biology
2017-2019
Centre for Addiction and Mental Health
2015-2018
Medical Research Council
2017
Toronto Western Hospital
2015
University Health Network
2015
A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (CRISPR-based with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, date, have been used at limited scales. Here, we perform genome-scale targeting all expressed genes CRISPR interference (CRISPRi) across >2.5 million human cells. We use transcriptional phenotypes predict function poorly characterized...
A new way into the ER Membrane-embedded proteins are highly diverse in topology, physical characteristics, and location. This diversity necessitates multiple pathways for their effective membrane insertion. Guna et al. found that a widely conserved protein complex is responsible inserting subset of endoplasmic reticulum (ER) (see Perspective by Fry Clemons Jr.). (EMC) inserts transmembrane domains whose topology hydrophobicity preclude recognition other insertion factors. finding helps...
Mammals encode ∼5,000 integral membrane proteins that need to be inserted in a defined topology at the endoplasmic reticulum (ER) by mechanisms are incompletely understood. Here, we found efficient biogenesis of β1-adrenergic receptor (β1AR) and other G protein-coupled receptors (GPCRs) requires conserved ER protein complex (EMC). Reconstitution studies β1AR narrowed EMC requirement co-translational insertion first transmembrane domain (TMD). Without EMC, proportion TMD1 an inverted...
In the mitochondrial outer membrane, α-helical transmembrane proteins play critical roles in cytoplasmic-mitochondrial communication. Using genome-wide CRISPR screens, we identified carrier homolog 2 (MTCH2), and its paralog MTCH1, showed that it is required for insertion of biophysically diverse tail-anchored (TA), signal-anchored, multipass proteins, but not membrane β-barrel proteins. Purified MTCH2 was sufficient to mediate into reconstituted proteoliposomes. Functional mutational...
Abstract Optimum protein function and biochemical activity critically depends on water availability because solvent thermodynamics drive folding macromolecular interactions 1 . Reciprocally, macromolecules restrict the movement of ‘structured’ molecules within their hydration layers, reducing available ‘free’ bulk therefore total thermodynamic potential energy water, or potential. Here, concentrated solutions such as cytosol, we found that modest changes in temperature greatly affect...
22q11.2 deletion syndrome (22q11.2DS) is the most common micro-deletion syndrome. The associated conveys strongest known molecular risk for schizophrenia. Neurodevelopmental phenotypes, including intellectual disability, are also prominent though variable in severity. Other developmental features include congenital cardiac and craniofacial anomalies. Whereas existing mouse models have been helpful determining role of some genes overlapped by hemizygous phenotypic expression, much remains...
CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions cell biology, genetics, biotechnology, but wider deployment CRISPRi screening has been constrained by large size single guide RNA (sgRNA) libraries challenges generating models with consistent knockdown. Here, we present next-generation sgRNA effector...
The infection of mammalian cells by viruses and innate immune responses to are spatiotemporally organized processes. Cytosolic RNA sensors trigger nuclear translocation the transcription factor interferon regulatory 3 (IRF3) consequent induction host viruses. Previous genetic screens for factors involved in viral sensing did not resolve changes subcellular localization or proteins. Here, we increased throughput our optical pooled screening technology over fourfold. This allowed us carry out...
Background Genetic testing in psychiatry promises to improve patient care through advances personalised medicine. However, there are few clinically relevant examples. Aims To determine whether patients with a well-established genetic subtype of schizophrenia show different response profile the antipsychotic clozapine than those idiopathic schizophrenia. Method We retrospectively studied long-term safety and efficacy 40 adults schizophrenia, half 22q11.2 deletion (22q11.2DS group) matched for...
Clinical molecular testing has been available for 22q11.2 deletion syndrome (22q11.2DS) over two decades yet under‐recognition and diagnostic delays are common. To characterize the “diagnostic odyssey” in 22q11.2DS we studied 202 well‐characterized unrelated adults, none ascertained through an affected relative. We used a regression model to identify clinical demographic factors associated with length of time diagnosis. Kaplan–Meier analysis compared diagnosis era (since 1994) earlier birth...
Between 6-20% of the cellular proteome is under circadian control and tunes mammalian cell function with daily environmental cycles. For viability, to maintain volume within narrow limits, variation in osmotic potential exerted by changes soluble must be counterbalanced. The mechanisms consequences this compensation have not been investigated before. In cultured cells tissue we find that involves electroneutral active transport Na+, K+, Cl- through differential activity SLC12A family...
ABSTRACT Translation of mRNAs containing premature termination codons (PTCs) results in truncated protein products with deleterious effects. Nonsense-mediated decay (NMD) is a surveillance pathway responsible for detecting PTC transcripts. Although the molecular mechanisms governing mRNA degradation have been extensively studied, fate nascent product remains largely uncharacterized. Here, we use fluorescent reporter system mammalian cells to reveal selective specifically targeting an NMD...
Mitochondrial outer membrane ⍺-helical proteins play critical roles in mitochondrial-cytoplasmic communication, but the rules governing targeting and insertion of these biophysically diverse remain unknown. Here, we first defined complement required mammalian biogenesis machinery through genome-wide CRISPRi screens using topologically distinct proteins. Systematic analysis nine identified factors across 21 substrates reveals that components are organized into pathways act on based their...
Abstract A central goal of genetics is to define the relationships between genotypes and phenotypes. High-content phenotypic screens such as Perturb-seq (pooled CRISPR-based with single-cell RNA-sequencing readouts) enable massively parallel functional genomic mapping but, date, have been used at limited scales. Here, we perform genome-scale targeting all expressed genes CRISPR interference (CRISPRi) across >2.5 million human cells present a framework power biological discovery resulting...
A large proportion of membrane proteins must be assembled into oligomeric complexes for function. How this process occurs is poorly understood, but it clear that complex assembly tightly regulated to avoid accumulation orphan subunits with potential cytotoxic effects. We interrogated in mammalian cells by using the WRB/CAML complex, an essential insertase tail-anchored endoplasmic reticulum (ER), as a model system. Our data suggest stability each subunit differentially regulated. In WRB's...
Abstract Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, query epistatic relationships at scale. This enabled by flexible dual-sgRNA library design that allows the simultaneous delivery selection of fixed sgRNA second randomized guide, comprised genome-wide library, single...
Abstract CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions cell biology, genetics, biotechnology, but wider deployment CRISPRi screening has been constrained by large size single guide RNA (sgRNA) libraries challenges generating models with consistent knockdown. Here, we present next-generation sgRNA...
Mapping genetic interactions is essential for determining gene function and defining novel biological pathways. We report a simple to use CRISPR interference (CRISPRi) based platform, compatible with Fluorescence Activated Cell Sorting (FACS)-based reporter screens, query epistatic relationships at scale. This enabled by flexible dual-sgRNA library design that allows the simultaneous delivery selection of fixed sgRNA second randomized guide, comprised genome-wide library, single...
Mammalian membrane proteins perform essential physiologic functions that rely on their accurate insertion and folding at the endoplasmic reticulum (ER). Using forward arrayed genetic screens, we systematically studied biogenesis of a panel proteins, including several G-protein coupled receptors (GPCRs). We observed central role for insertase, ER protein complex (EMC), developed dual-guide approach to identify modifiers EMC. found back sec61 (BOS) complex, component 'multipass translocon',...