New factors for protein transport identified by a genome-wide CRISPRi screen in mammalian cells

Protein and membrane trafficking pathways are critical for cell and tissue homeostasis. Traditional genetic and biochemical approaches have shed light on basic principles underlying these processes. However, the list of factors required for secretory pathway function remains incomplete, and mechanisms involved in their adaptation poorly understood. Here, we present a powerful strategy based on a pooled genome-wide CRISPRi screen that allowed the identification of new factors involved in protein transport. Two newly identified factors, TTC17 and CCDC157, localized along the secretory pathway and were found to interact with resident proteins of ER-Golgi membranes. In addition, we uncovered that upon TTC17 knockdown, the polarized organization of Golgi cisternae was altered, creating glycosylation defects, and that CCDC157 is an important factor for the fusion of transport carriers to Golgi membranes. In conclusion, our work identified and characterized new actors in the mechanisms of protein transport and secretion, and opens stimulating perspectives for the use of our platform in physiological and pathological contexts.Includes Wellcome Trust, MRC and H202

Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening

Forward genetic screens are powerful tools for the unbiased discovery and functional characterization of specific genetic elements associated with a phenotype of interest. Recently, the RNA-guided endonuclease Cas9 from the microbial CRISPR (clustered regularly interspaced short palindromic repeats) immune system has been adapted for genome-scale screening by combining Cas9 with pooled guide RNA libraries. Here we describe a protocol for genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system. Custom- or ready-made guide RNA libraries are constructed and packaged into lentiviral vectors for delivery into cells for screening. As each screen is unique, we provide guidelines for determining screening parameters and maintaining sufficient coverage. To validate candidate genes identified by the screen, we further describe strategies for confirming the screening phenotype, as well as genetic perturbation, through analysis of indel rate and transcriptional activation. Beginning with library design, a genome-scale screen can be completed in 9-15 weeks, followed by 4-5 weeks of validation.Paul & Daisy Soros Fellowships for New Americans (New York, N.Y.)McGovern Institute for Brain Research at MIT (Friends of McGovern Institute Fellowship)Massachusetts Institute of Technology. Poitras Center for Affective Disorders ResearchUnited States. Department of Energy (Computational Science Graduate Fellowship)National Institute of Mental Health (U.S.) (5DP1-MH100706)National Institute of Mental Health (U.S.) (1R01-MH110049)New York Stem Cell FoundationPoitras FoundationSimons FoundationPaul G. Allen Family FoundationVallee FoundationTom HarrimanB. Metcalf

Genome-wide association studies of cancer: current insights and future perspectives.

Genome-wide association studies (GWAS) provide an agnostic approach for investigating the genetic basis of complex diseases. In oncology, GWAS of nearly all common malignancies have been performed, and over 450 genetic variants associated with increased risks have been identified. As well as revealing novel pathways important in carcinogenesis, these studies have shown that common genetic variation contributes substantially to the heritable risk of many common cancers. The clinical application of GWAS is starting to provide opportunities for drug discovery and repositioning as well as for cancer prevention. However, deciphering the functional and biological basis of associations is challenging and is in part a barrier to fully unlocking the potential of GWAS

Formal Verification of the CAA-Design of the Production Cell

Coordinated atomic actions (CAAs) have been used in a semi-formal way for the design of the production cell case study. This paper presents a formal specification and verification of the production cell building on this design. Each CAA is modelled as an atomic state transition characterized by its pre- and postconditions. In order for such transitions to become enabled, conditions are formalized requiring all associated roles to be activated. Activation of roles is performed by controllers, which are again modelled as state transitions. The state space of the production cell can be viewed as being finite; hence, the production cell is specified as a finite state transition system and the formal verification of the CAA-design is carried out using model-checking. 1 Introduction Coordinated atomic actions (CAA) [RRS + 97, XRR + 95] have been proposed as a concept for structuring complex concurrent activities in safety-critical systems. The principles of applying CAAs to the design of..

Formal Specification and Verification of Object-Based Systems in a Temporal Logic Setting

This paper presents an approach to specification, refinement and verification of object-based systems in a temporal logic framework. The behaviour of an objectbased system is viewed as derivable from the behaviours of its constituent component objects. Temporal logic is a formalism well suited for specifying behaviour of concurrent systems; it also provides conceptually simple notions of composition and refinement: Composition of objects is expressed as conjunction of the associated component specifications. The refinement relation between a low-level and a high-level specification requires that the former specification implies the latter. Specifically in an object-based approach, systems and their components need to be viewed as open systems: Each object guarantees some service (behaviour), provided its environment conforms to certain assumptions. Hence, such components are most appropriately specified in an assumption/guarantee style. The approach presented here adopts TLA as the und..