Perturb-Seq: Dissecting Molecular Circuits with Scalable Single-Cell RNA Profiling of Pooled Genetic Screens

Genetic screens help infer gene function in mammalian cells, but it has remained difficult to assay complex phenotypes—such as transcriptional profiles—at scale. Here, we develop Perturb-seq, combining single-cell RNA sequencing (RNA-seq) and clustered regularly interspaced short palindromic repeats (CRISPR)-based perturbations to perform many such assays in a pool. We demonstrate Perturb-seq by analyzing 200,000 cells in immune cells and cell lines, focusing on transcription factors regulating the response of dendritic cells to lipopolysaccharide (LPS). Perturb-seq accurately identifies individual gene targets, gene signatures, and cell states affected by individual perturbations and their genetic interactions. We posit new functions for regulators of differentiation, the anti-viral response, and mitochondrial function during immune activation. By decomposing many high content measurements into the effects of perturbations, their interactions, and diverse cell metadata, Perturb-seq dramatically increases the scope of pooled genomic assays. Keywords: single-cell RNA-seq; pooled screen; CRISPR; epistasis; genetic interaction

Tradespace Investigation of a Telescope Architecture for Next-generation Space Astronomy and Exploration

Humanity’s endeavor to further its scientific understanding of the celestial heavens has led to the creation and evolution of increasingly powerful and complex space telescopes. Space telescopes provide a view of the solar system, galaxy, and universe unobstructed by Earth’s atmosphere and have profoundly changed the way people view space. In an effort to further advance space telescope capability and achieve the accompanying scientific understanding, the Massachusetts Institute of Technology (MIT), specifically, course 16.89 Space Systems Engineering, explored the tradespace of architectural enumerations encompassed within the design of an ultraviolet-optical-infrared (UVOIR) space telescope located at Sun-Earth Lagrangian Point Two (SE-L2). SE-L2 presents several advantages as an operating location for a UVOIR telescope such as a thermally stable environment and an orbit that allows the telescope to maintain a constant orientation with respect to all of the primary sources of heat and light. The main disadvantages associated with SE-L2 are caused by its relatively large distance from Earth, which marginalizes the effectiveness of real-time telerobotics because of latency and increases the cost of communications, launch, and servicing. Course 16.89 believes that, for this UVOIR application, the strengths of this operating location outweigh its weaknesses and therefore decided to explore the family of opportunities associated with SE-L2. This course used appropriate performance and system metrics to quantify the effectiveness of the aforementioned architectures and create a Pareto front of viable architectures. Evaluating the designs along the Pareto front allowed the course to characterize and group architectures and present these group-types to stakeholders for the selection of an optimal space telescope according to stakeholder requirements and resources. This course also developed sensitivity analysis, which allowed for a greater understanding of how architectural decisions affect the performance of the satellite. Segmentation, modularity, assembly, autonomy, and servicing were key aspects of this multidimensional analysis given the 16.8-meter class size and location of the telescope. Within the respective operating environment and for a spacecraft of similar characteristics, this model will allow stakeholders to predict the long-term operational effectiveness of different space telescope architectures and capture the synergistic effects of combining various architectural decisions into a spacecraft design. The following sections step through the aforesaid analysis and design efforts conducted in 16.89 beginning with Section III, which explicitly performs the stakeholder analysis and articulates the requirements of the mission. Section IV gives an overview of past designs and expands upon the architecture enumerations pertinent to this project, while Section V presents the methods and metrics by which those architectures will be evaluated and the system metrics which will be balanced and optimized in the creation of this space telescope. Section VI will present the model validation of this project and Section VII will discuss the results and analyses of the project. Finally, Section VIII will explore the future work opportunities of this project, while Section IX will present the conclusions and recommendations drawn from this project.MIT Department of Aeronautics and Astronautic

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

Methods for bounding genetic nonlinearities

Thesis: Ph. D. in Medical Engineering and Medical Physics, Harvard-MIT Program in Health Sciences and Technology, 2018.Cataloged from PDF version of thesis.Includes bibliographical references.Complex hierarchical structures are a hallmark of life. Within multicellular organisms, the building blocks of these structures are cells; within cells, they are genes. The interdependence of these building blocks is difficult to measure but is integral to the biological processes of health and disease, which emerge from the dynamism of thousands of interacting genes. This cooperativity manifests in particular mutations which accumulate over the course of cancer progression, gender-specific medical conditions, and transcription factor cocktails used to reprogram differentiated cells into stem cells. However, it is experimentally intractable to test the significance of perturbing every unique combination of genes. Instead, we explore gross features of this interaction space to determine how prevalent these synergies are. We take a top-down approach, creating new methods to measure the effects of removing genes from the full set. In the first, we develop a method to measure the transcriptional response to genetic perturbations across hundreds of thousands of cells revealing opposing classes of transcription factors regulating the immune response of dendritic cells. In the second, we create a method to measure how millions of combinations of genetic perturbations impact the growth rate of cancer cell lines.by Atray Dixit.Ph. D. in Medical Engineering and Medical Physic

Correction: Anastopoulos et al. Multi-Drug Featurization and Deep Learning Improve Patient-Specific Predictions of Adverse Events. Int. J. Environ. Res. Public Health 2021, 18, 2600

In the original publication [...

Single-cell RNA-seq reveals changes in cell cycle and differentiation programs upon aging of hematopoietic stem cells

Both intrinsic cell state changes and variations in the composition of stem cell populations have been implicated as contributors to aging. We used single-cell RNA-seq to dissect variability in hematopoietic stem cell (HSC) and hematopoietic progenitor cell populations from young and old mice from two strains. We found that cell cycle dominates the variability within each population and that there is a lower frequency of cells in the G1 phase among old compared with young long-term HSCs, suggesting that they traverse through G1 faster. Moreover, transcriptional changes in HSCs during aging are inversely related to those upon HSC differentiation, such that old short-term (ST) HSCs resemble young long-term (LT-HSCs), suggesting that they exist in a less differentiated state. Our results indicate both compositional changes and intrinsic, population-wide changes with age and are consistent with a model where a relationship between cell cycle progression and self-renewal versus differentiation of HSCs is affected by aging and may contribute to the functional decline of old HSCs.Howard Hughes Medical InstituteNational Institutes of Health (U.S.) (Centers of Excellence in Genomic Science Award 1P50HG006193-01)National Cancer Institute (U.S.) (Koch Institute Support (Core) Grant P30-CA14051)Klarman Cell Observator

A Genome-wide CRISPR Screen in Primary Immune Cells to Dissect Regulatory Networks

Finding the components of cellular circuits and determining their functions systematically remains a major challenge in mammalian cells. Here, we introduced genome-wide pooled CRISPR-Cas9 libraries into dendritic cells (DCs) to identify genes that control the induction of tumor necrosis factor (Tnf) by bacterial lipopolysaccharide (LPS), a key process in the host response to pathogens, mediated by the Tlr4 pathway. We found many of the known regulators of Tlr4 signaling, as well as dozens of previously unknown candidates that we validated. By measuring protein markers and mRNA profiles in DCs that are deficient in known or candidate genes, we classified the genes into three functional modules with distinct effects on the canonical responses to LPS and highlighted functions for the PAF complex and oligosaccharyltransferase (OST) complex. Our findings uncover new facets of innate immune circuits in primary cells and provide a genetic approach for dissection of mammalian cell circuits.Broad InstituteNational Institutes of Health (U.S.) (NIMH: 5DP1-MH100706)National Institutes of Health (U.S.) (NIDDK: 5R01-DK097768)National Science Foundation (U.S.) (Waterman Award)W. M. Keck FoundationNew York Stem Cell FoundationDamon Runyon Cancer Research FoundationSearle Scholars ProgramVallee FoundationRobert MetcalfeMassachusetts Institute of Technology. Simons Center for the Social BrainNational Human Genome Research Institute (U.S.) (K99- HG008171)National Science Foundation (U.S.). Graduate Research Fellowship Program (grant number 1122374)Human Frontier Science Program (Strasbourg, France