SUMMARY 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 ~1000-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, retro-translocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways.

We recently found that nucleosomes directly block access of CRISPR/Cas9 to DNA (Horlbeck et al., 2016). Here, we build on this observation with a comprehensive algorithm that incorporates chromatin, position, and sequence features to accurately predict highly effective single guide RNAs (sgRNAs) for targeting nuclease-dead Cas9-mediated transcriptional repression (CRISPRi) and activation (CRISPRa). We use this algorithm to design next-generation genome-scale CRISPRi and CRISPRa libraries targeting human and mouse genomes. A CRISPRi screen for essential genes in K562 cells demonstrates that the large majority of sgRNAs are highly active. We also find CRISPRi does not exhibit any detectable non-specific toxicity recently observed with CRISPR nuclease approaches. Precision-recall analysis shows that we detect over 90% of essential genes with minimal false positives using a compact 5 sgRNA/gene library. Our results establish CRISPRi and CRISPRa as premier tools for loss- or gain-of-function studies and provide a general strategy for identifying Cas9 target sites.DOI: http://dx.doi.org/10.7554/eLife.19760.001

SUMMARY Genetic interaction (GI) maps, comprising pairwise measures of how strongly the function of one gene depends on the presence of a second, have enabled the systematic exploration of gene function in microorganisms. Here, we present a two-stage strategy to construct high-density GI maps in mammalian cells. First, we use ultra-complex pooled shRNA libraries (25 shRNAs/gene) to identify high-confidence hit genes for a given phenotype and effective shRNAs. We then construct double-shRNA libraries from these to systematically measure GIs between hits. A GI map focused on ricin susceptibility broadly recapitulates known pathways and provides many unexpected insights. These include a non-canonical role for COPI, a novel protein complex (SRIC) affecting toxin clearance, a specialized role for the ribosomal protein RPS25, and functionally distinct mammalian TRAPP complexes. The ability to rapidly generate mammalian GI maps provides a potentially transformative tool for defining gene function and designing combination therapies based on synergistic pairs.

Summary p97 is a AAA-ATPase with multiple cellular functions, one of which is critical regulation of protein homeostasis pathways. We describe the characterization of CB-5083, a potent, selective and orally bioavailable inhibitor of p97. Treatment of tumor cells with CB-5083 leads to accumulation of poly-ubiquitinated proteins, retention of endoplasmic reticulum associated degradation (ERAD) substrates and generation of irresolvable proteotoxic stress leading to activation of the apoptotic arm of the unfolded protein response (UPR). In xenograft models, CB-5083 causes modulation of key p97-related pathways, induces apoptosis and has antitumor activity in a broad range of both hematological and solid tumor models. Molecular determinants of CB-5083 activity include expression of genes in the ERAD pathway providing a potential strategy for patient selection.

Highlights d A CRISPR interference platform for genetic screens in human iPSC-derived neurons d Survival screens uncover genes essential for neurons, but not iPSCs or cancer cells d Single-cell RNA-seq screens reveal distinct neuronal roles for ubiquitous genes d Arrayed high-content screens uncover genes controlling neuronal morphology

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Alzheimer’s disease (AD) is characterized by the selective vulnerability of specific neuronal populations, the molecular signatures of which are largely unknown. To identify and characterize selectively vulnerable neuronal populations, we used single-nucleus RNA sequencing to profile the caudal entorhinal cortex and the superior frontal gyrus – brain regions where neurofibrillary inclusions and neuronal loss occur early and late in AD, respectively – from postmortem brains spanning the progression of AD-type tau neurofibrillary pathology. We identified RORB as a marker of selectively vulnerable excitatory neurons in the entorhinal cortex, and subsequently validated their depletion and selective susceptibility to neurofibrillary inclusions during disease progression using quantitative neuropathological methods. We also discovered an astrocyte subpopulation, likely representing reactive astrocytes, characterized by decreased expression of genes involved in homeostatic functions. Our characterization of selectively vulnerable neurons in AD paves the way for future mechanistic studies of selective vulnerability and potential therapeutic strategies for enhancing neuronal resilience.

Summary The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau (MAPT) plays a central role in the pathogenesis of several forms of dementia known as tauopathies, including Alzheimer’s disease (AD), frontotemporal dementia (FTD) and chronic traumatic encephalopathy (CTE) 1 . Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity 2 . This observation and complementary experimental studies 3 , 4 have suggested that tau can spread in a prion-like manner by passing to naïve cells where it templates misfolding and aggregation. However, while tau propagation has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein (LDL) receptor-related protein 1 (LRP1) controls tau endocytosis and subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and iPS-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule binding repeat region of tau. Furthermore, we find that downregulation of LRP1 in an in vivo mouse model of tau spread effectively reduced tau propagation between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain and, thus, as a novel target for diseases of tau spread and aggregation.