43 research outputs found
Versatile in vivo regulation of tumor phenotypes by dCas9-mediated transcriptional perturbation
Targeted transcriptional regulation is a powerful tool to study genetic mediators of cellular behavior. Here, we show that catalytically dead Cas9 (dCas9) targeted to genomic regions upstream or downstream of the transcription start site allows for specific and sustainable gene-expression level alterations in tumor cells in vitro and in syngeneic immune-competent mouse models. We used this approach for a high-coverage pooled gene-activation screen in vivo and discovered previously unidentified modulators of tumor growth and therapeutic response. Moreover, by using dCas9 linked to an activation domain, we can either enhance or suppress target gene expression simply by changing the genetic location of dCas9 binding relative to the transcription start site. We demonstrate that these directed changes in gene-transcription levels occur with minimal off-target effects. Our findings highlight the use of dCas9-mediated transcriptional regulation as a versatile tool to reproducibly interrogate tumor phenotypes in vivo. Keywords: cancer genetics; cancer models; cancer therapeutic resistance; gene regulation; CRISPRNational Cancer Institute (U.S.) (Grant U54-CA112967-06)National Cancer Institute (U.S.) (Grant P30-CA14051
Identification of a transporter complex responsible for the cytosolic entry of nitrogen-containing bisphosphonates
Nitrogen-containing-bisphosphonates (N-BPs) are widely prescribed to treat osteoporosis and other bone-related diseases. Although previous studies established that N-BPs function by inhibiting the mevalonate pathway in osteoclasts, the mechanism by which N-BPs enter the cytosol from the extracellular space to reach their molecular target is not understood. Here we implemented a CRISPRi-mediated genome-wide screen and identified SLC37A3 (solute carrier family 37 member A3) as a gene required for the action of N-BPs in mammalian cells. We observed that SLC37A3 forms a complex with ATRAID (all-trans retinoic acid-induced differentiation factor), a previously identified genetic target of N-BPs. SLC37A3 and ATRAID localize to lysosomes and are required for releasing N-BP molecules that have trafficked to lysosomes through fluid-phase endocytosis into the cytosol. Our results elucidate the route by which N-BPs are delivered to their molecular target, addressing a key aspect of the mechanism of action of N-BPs that may have significant clinical relevance
Single-cell analysis of long non-coding RNAs in the developing human neocortex
Single cell transcriptomics of lncRNA expression in K562 cell cultures. A Distributions of median lncRNA expression to median mRNA expression ratios (lncRNA:mRNA) in populations, in silico merged single cells, and single cells from K562 cultures. B Proportion of K562 cells that expressed each lncRNA (blue) and mRNA (red), separated by maximum expression in single cells. C Same as in (B) but grouped by maximum expression quantile. D Distributions of non-zero lncRNA (blue) and mRNA (red) expression in 46 single K562 cells. Green squares, housekeeping genes; black triangles, ERCC Spike-In Controls. (PDF 454 kb
Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation
While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%–99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a ∼1,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways
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CRISPRi-based radiation modifier screen identifies long non-coding RNA therapeutic targets in glioma.
BackgroundLong non-coding RNAs (lncRNAs) exhibit highly cell type-specific expression and function, making this class of transcript attractive for targeted cancer therapy. However, the vast majority of lncRNAs have not been tested as potential therapeutic targets, particularly in the context of currently used cancer treatments. Malignant glioma is rapidly fatal, and ionizing radiation is part of the current standard-of-care used to slow tumor growth in both adult and pediatric patients.ResultsWe use CRISPR interference (CRISPRi) to screen 5689 lncRNA loci in human glioblastoma (GBM) cells, identifying 467 hits that modify cell growth in the presence of clinically relevant doses of fractionated radiation. Thirty-three of these lncRNA hits sensitize cells to radiation, and based on their expression in adult and pediatric gliomas, nine of these hits are prioritized as lncRNA Glioma Radiation Sensitizers (lncGRS). Knockdown of lncGRS-1, a primate-conserved, nuclear-enriched lncRNA, inhibits the growth and proliferation of primary adult and pediatric glioma cells, but not the viability of normal brain cells. Using human brain organoids comprised of mature neural cell types as a three-dimensional tissue substrate to model the invasive growth of glioma, we find that antisense oligonucleotides targeting lncGRS-1 selectively decrease tumor growth and sensitize glioma cells to radiation therapy.ConclusionsThese studies identify lncGRS-1 as a glioma-specific therapeutic target and establish a generalizable approach to rapidly identify novel therapeutic targets in the vast non-coding genome to enhance radiation therapy
A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response
Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.National Human Genome Research Institute (U.S.) (Grant P50HG006193
Genome-scale discovery of protein-coding and lncRNA gene function with CRISPRi and CRISPRa
The genome sequencing efforts of the past decade have cataloged the universe of protein-coding and non-coding transcripts produced by the human genome. A central challenge now is to understand how these many thousands of genes act to mediate the vast array of cellular processes involved in either normal or disease states. To accomplish this, we require specific and scaleable tools to manipulate the expression of individual genes and measure their functional contribution in a given cellular context. In this thesis, I describe the initial development and subsequent refinement of genome-scale genetic screening technologies based on CRISPR-mediated interference and activation. I present the results of several screens for protein-coding genes that modulate cell proliferation and toxin susceptibility. Finally, I describe the application of CRISPR interference in the systematic repression of long non-coding RNA genes, a large class of genes of which very few genes have any known function. The screens described here identify nearly 500 lncRNA genes that modify cell growth in at least one of seven diverse cell lines, and furthermore highlight the exquisite cell type-specificity of lncRNA function
ER Cargo Properties Specify a Requirement for COPII Coat Rigidity Mediated by Sec13p
Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated bythe essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31,forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst)mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPIIcoat and the cellular context in which it could still function. Genetic and biochemical analysessuggested that Sec13p was required to generate vesicles from membranes that contained asymmetricallydistributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify theCOPII cage and increase its membrane-bending capacity; this function could be bypassed when a bstmutation renders the membrane more deformable