Combinatorial CRISPR-Cas9 screens for de novo mapping of genetic interactions.

We developed a systematic approach to map human genetic networks by combinatorial CRISPR-Cas9 perturbations coupled to robust analysis of growth kinetics. We targeted all pairs of 73 cancer genes with dual guide RNAs in three cell lines, comprising 141,912 tests of interaction. Numerous therapeutically relevant interactions were identified, and these patterns replicated with combinatorial drugs at 75% precision. From these results, we anticipate that cellular context will be critical to synthetic-lethal therapies

CRISPR-Cas9 screens in human cells and primary neurons identify modifiers of C9ORF72 dipeptide-repeat-protein toxicity.

Hexanucleotide-repeat expansions in the C9ORF72 gene are the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (c9ALS/FTD). The nucleotide-repeat expansions are translated into dipeptide-repeat (DPR) proteins, which are aggregation prone and may contribute to neurodegeneration. We used the CRISPR-Cas9 system to perform genome-wide gene-knockout screens for suppressors and enhancers of C9ORF72 DPR toxicity in human cells. We validated hits by performing secondary CRISPR-Cas9 screens in primary mouse neurons. We uncovered potent modifiers of DPR toxicity whose gene products function in nucleocytoplasmic transport, the endoplasmic reticulum (ER), proteasome, RNA-processing pathways, and chromatin modification. One modifier, TMX2, modulated the ER-stress signature elicited by C9ORF72 DPRs in neurons and improved survival of human induced motor neurons from patients with C9ORF72 ALS. Together, our results demonstrate the promise of CRISPR-Cas9 screens in defining mechanisms of neurodegenerative diseases

An In Vitro Model for Lewy Body-Like Hyaline Inclusion/Astrocytic Hyaline Inclusion: Induction by ER Stress with an ALS-Linked SOD1 Mutation

Neuronal Lewy body-like hyaline inclusions (LBHI) and astrocytic hyaline inclusions (Ast-HI) containing mutant Cu/Zn superoxide dismutase 1 (SOD1) are morphological hallmarks of familial amyotrophic lateral sclerosis (FALS) associated with mutant SOD1. However, the mechanisms by which mutant SOD1 contributes to formation of LBHI/Ast-HI in FALS remain poorly defined. Here, we report induction of LBHI/Ast-HI-like hyaline inclusions (LHIs) in vitro by ER stress in neuroblastoma cells. These LHI closely resemble LBHI/Ast-HI in patients with SOD1-linked FALS. LHI and LBHI/Ast-HI share the following features: 1) eosinophilic staining with a pale core, 2) SOD1, ubiquitin and ER resident protein (KDEL) positivity and 3) the presence of approximately 15–25 nm granule-coated fibrils, which are morphological hallmark of mutant SOD1-linked FALS. Moreover, in spinal cord neurons of L84V SOD1 transgenic mice at presymptomatic stage, we observed aberrant aggregation of ER and numerous free ribosomes associated with abnormal inclusion-like structures, presumably early stage neuronal LBHI. We conclude that the LBHI/Ast-HI seen in human patients with mutant SOD1-linked FALS may arise from ER dysfunction

A genome-wide CRISPR screen identifies a restricted set of HIV host dependency factors

Host proteins are essential for HIV entry and replication and can be important nonviral therapeutic targets. Large-scale RNA interference (RNAi)-based screens have identified nearly a thousand candidate host factors, but there is little agreement among studies and few factors have been validated. Here we demonstrate that a genome-wide CRISPR-based screen identifies host factors in a physiologically relevant cell system. We identify five factors, including the HIV co-receptors CD4 and CCR5, that are required for HIV infection yet are dispensable for cellular proliferation and viability. Tyrosylprotein sulfotransferase 2 (TPST2) and solute carrier family 35 member B2 (SLC35B2) function in a common pathway to sulfate CCR5 on extracellular tyrosine residues, facilitating CCR5 recognition by the HIV envelope. Activated leukocyte cell adhesion molecule (ALCAM) mediates cell aggregation, which is required for cell-to-cell HIV transmission. We validated these pathways in primary human CD4 + T cells through Cas9-mediated knockout and antibody blockade. Our findings indicate that HIV infection and replication rely on a limited set of host-dispensable genes and suggest that these pathways can be studied for therapeutic intervention

Morphing in nature and beyond: a review of natural and synthetic shape-changing materials and mechanisms

Shape-changing materials open an entirely new solution space for a wide range of disciplines: from architecture that responds to the environment and medical devices that unpack inside the body, to passive sensors and novel robotic actuators. While synthetic shape-changing materials are still in their infancy, studies of biological morphing materials have revealed key paradigms and features which underlie efficient natural shape-change. Here, we review some of these insights and how they have been, or may be, translated to artificial solutions. We focus on soft matter due to its prevalence in nature, compatibility with users and potential for novel design. Initially, we review examples of natural shape-changing materials—skeletal muscle, tendons and plant tissues—and compare with synthetic examples with similar methods of operation. Stimuli to motion are outlined in general principle, with examples of their use and potential in manufactured systems. Anisotropy is identified as a crucial element in directing shape-change to fulfil designed tasks, and some manufacturing routes to its achievement are highlighted. We conclude with potential directions for future work, including the simultaneous development of materials and manufacturing techniques and the hierarchical combination of effects at multiple length scales.</p

Weak base pairing in both seed and 3' regions reduces RNAi off-targets and enhances si/shRNA designs

© The Author(s) 2014.The use of RNA interference is becoming routine in scientific discovery and treatment of human disease. However, its applications are hampered by unwanted effects, particularly off-targeting through miRNA-like pathways. Recent studie

Next-generation libraries for robust RNA interference-based genome-wide screens

Genetic screening based on loss-of-function phenotypes is a powerful discovery tool in biology. Although the recent development of clustered regularly interspaced short palindromic repeats (CRISPR)-based screening approaches in mammalian cell culture has enormous potential, RNA interference (RNAi)-based screening remains the method of choice in several biological contexts. We previously demonstrated that ultracomplex pooled short-hairpin RNA (shRNA) libraries can largely overcome the problem of RNAi off-target effects in genome-wide screens. Here, we systematically optimize several aspects of our shRNA library, including the promoter and microRNA context for shRNA expression, selection of guide strands, and features relevant for postscreen sample preparation for deep sequencing. We present next-generation high-complexity libraries targeting human and mouse protein-coding genes, which we grouped into 12 sublibraries based on biological function. A pilot screen suggests that our next-generation RNAi library performs comparably to current CRISPR interference (CRISPRi)-based approaches and can yield complementary results with high sensitivity and high specificity131351sciescopu