A5060001686- CRISPR and Genetic Engineering
- Porphyrin Metabolism and Disorders
- Single-cell and spatial transcriptomics
- Folate and B Vitamins Research
- Enzyme Structure and Function
- Metabolism and Genetic Disorders
- DNA Repair Mechanisms
- Angiogenesis and VEGF in Cancer
- Cell Image Analysis Techniques
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- RNA modifications and cancer
- Bacterial Genetics and Biotechnology
- Microtubule and mitosis dynamics
- Photosynthetic Processes and Mechanisms
- DNA and Nucleic Acid Chemistry
- Peptidase Inhibition and Analysis
- Ubiquitin and proteasome pathways
- Protease and Inhibitor Mechanisms
- Metalloenzymes and iron-sulfur proteins
- Trace Elements in Health
- Evolution and Genetic Dynamics
- Biochemical and Molecular Research
- RNA Interference and Gene Delivery
- Adipose Tissue and Metabolism
Harvard University
2021-2024
Boston VA Research Institute
2024
Massachusetts Institute of Technology
2012-2023
Whitehead Institute for Biomedical Research
2022-2023
University of California, San Francisco
2016-2022
QB3
2016-2022
Howard Hughes Medical Institute
2015-2021
University of San Francisco
2021
IIT@MIT
2015
University of Freiburg
2003-2010
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...
Manifold destiny Mapping of genetic interactions (GIs) is usually based on cell fitness as the phenotypic readout, which obscures mechanistic origin interactions. Norman et al. developed a framework for mapping and understanding GIs. This approach leverages high-dimensional single-cell RNA sequencing data gathered from CRISPR-mediated, pooled perturbation screens. Diverse transcriptomic phenotypes construct “manifold” representing all possible states cell. Each GI projects state to...
Current single-cell RNA-sequencing approaches have limitations that stem from the microfluidic devices or fluid handling steps required for sample processing. We develop a method does not require specialized devices, expertise hardware. Our approach is based on particle-templated emulsification, which allows encapsulation and barcoding of cDNA in uniform droplet emulsions with only vortexer. Particle-templated instant partition sequencing (PIP-seq) accommodates wide range emulsification...
Abstract It has been suggested that monocytes/macrophages represent the pivotal cell type during early adaptive growth of pre-existent arterial anastomoses toward functional collateral arteries (arteriogenesis) upon occlusion. This hypothesis was supported by previous studies providing evidence elevation peripheral monocyte count, increased survival (e.g., granulocyte macrophage-colony stimulating factor), as well enhanced attraction or adhesion chemoattractant protein 1; intercellular...
Essential genes are the hubs of cellular networks, but lack high-throughput methods for titrating gene expression has limited our understanding fitness landscapes against which their levels optimized. We developed a modified CRISPRi system leveraging predictable reduction in efficacy imperfectly matched sgRNAs to generate defined activity and demonstrated its broad applicability. Using libraries mismatched predicted span full range knockdown levels, we characterized expression-fitness...
CRISPR (clustered regularly interspaced short palindromic repeats)-based gene inactivation provides a powerful means for linking genes to particular cellular phenotypes. CRISPR-based screening typically uses large genomic pools of single guide RNAs (sgRNAs). However, this approach is limited phenotypes that can be enriched by chemical selection or FACS sorting. Here, we developed microscopy-based approach, which name optical enrichment, select cells displaying CRISPR-induced phenotype...
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...
Monocyte adhesion to shear stress-activated endothelium stands as an important initial step during arteriogenesis (collateral artery growth). Using multiple approaches, we tested the hypothesis that monocyte via intercellular molecule-1 (ICAM-1) and selectin interactions is essential for adaptive arteriogenesis. Forty-eight New Zealand White rabbits received either solvent, chemoattractant protein-1 (MCP-1) alone, MCP-1 plus ICAM-mab, or IgG2a isotype control osmotic minipumps. After 7 days,...
Fungal indole prenyltransferases participate in a multitude of biosynthetic pathways. Their ability to prenylate diverse substrates has attracted interest for potential use chemoenzymatic synthesis. The fungal prenyltransferase FtmPT1 catalyzes the prenylation brevianamide F biosynthesis fumitremorgin-type alkaloids, which show pharmacological activities and are promising candidates development antitumor agents. Here, we report crystal structures unliganded Aspergillus fumigatus as well...
Polyhydroxybutyrate synthase (PhaC) catalyzes the polymerization of 3-(R)-hydroxybutyryl-coenzyme A as a means carbon storage in many bacteria. The resulting polymers can be used to make biodegradable materials with properties similar those thermoplastics and are an environmentally friendly alternative traditional petroleum-based plastics. full biochemical mechanistic understanding this process has been hindered part by lack structural information on PhaC. Here we present first structure...
The newly discovered light-dependent transcription factor CarH uses adenosylcobalamin as a light sensor to regulate expression of protective genes in bacteria upon exposure sunlight. This use is clever adaptation classic enzyme cofactor, taking advantage its photolabile Co–C bond. However, it also puzzling that photolysis generates the 5′-deoxyadenosyl radical could damage DNA. Here, using liquid chromatography and spectroscopic techniques, we demonstrate suppresses release instead effects...
5-Methylcytosine (5mC) in DNA CpG islands is an important epigenetic biomarker for mammalian gene regulation. It oxidized to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) by the ten-eleven translocation (TET) family enzymes, which are α-ketoglutarate (α-KG)/Fe(II)-dependent dioxygenases. In this work, we demonstrate that marker 5mC modified 5hmC, 5fC, 5caC vitro another class of α-KG/Fe(II)-dependent proteins—the repair enzymes AlkB family, include...
Dysfunction of the mitochondrial electron transport chain (mETC) is a major cause human diseases. To identify determinants mETC function, we screened genome-wide CRISPRi library under oxidative metabolic conditions with selective inhibition Complex III and identified ovarian carcinoma immunoreactive antigen (OCIA) domain-containing protein 1 (OCIAD1) as assembly factor. We find that OCIAD1 an inner membrane forms complex supramolecular prohibitin assemblies. Our data indicate required for...
GTPases are critical molecular switches involved in a wide range of biological functions. Recent phylogenetic and genomic analyses the large, mostly uncharacterized COG0523 subfamily revealed link between some proteins metal homeostasis pathways. In this report, we detail bioinorganic characterization YjiA, representative member subgroup 9 only protein to date with high-resolution structural information. We find that YjiA is capable binding several types transition metals dissociation...
The AlkB protein is a repair enzyme that uses an α-ketoglutarate/Fe(II)-dependent mechanism to alkyl DNA adducts. has been reported highly susceptible substrates, such as 1-methyladenine and 3-methylcytosine, more efficiently in ss-DNA than ds-DNA. Here, we tested the of weaker substrates 1-methylguanine 3-methylthymine found prefers them We also discovered its human homologues, ABH2 ABH3, are able aforementioned adducts when adduct present mismatched base pair. These observations...