Improved genome editing in human cell lines using the CRISPR method

The Cas9/CRISPR system has become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-introduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1

Rapid generation of endogenously driven transcriptional reporters in cells through CRISPR/Cas9

CRISPR/Cas9 technologies have been employed for genome editing to achieve gene knockouts and knock-ins in somatic cells. Similarly, certain endogenous genes have been tagged with fluorescent proteins. Often, the detection of tagged proteins requires high expression and sophisticated tools such as confocal microscopy and flow cytometry. Therefore, a simple, sensitive and robust transcriptional reporter system driven by endogenous promoter for studies into transcriptional regulation is desirable. We report a CRISPR/Cas9-based methodology for rapidly integrating a firefly luciferase gene in somatic cells under the control of endogenous promoter, using the TGFβ-responsive gene PAI-1. Our strategy employed a polycistronic cassette containing a non-fused GFP protein to ensure the detection of transgene delivery and rapid isolation of positive clones. We demonstrate that firefly luciferase cDNA can be efficiently delivered downstream of the promoter of the TGFβ-responsive gene PAI-1. Using chemical and genetic regulators of TGFβ signalling, we show that it mimics the transcriptional regulation of endogenous PAI-1 expression. Our unique approach has the potential to expedite studies on transcription of any gene in the context of its native chromatin landscape in somatic cells, allowing for robust high-throughput chemical and genetic screens

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

Repair of the TGFBI gene in human corneal keratocytes derived from a granular corneal dystrophy patient via CRISPR/Cas9-induced homology-directed repair

Abstract Granular corneal dystrophy (GCD) is an autosomal dominant hereditary disease in which multiple discrete and irregularly shaped granular opacities are deposited in the corneal stroma. GCD is caused by a point mutation in the transforming growth factor-β-induced (TGFBI) gene, located on chromosome 5q31. Here, we report the first successful application of CRISPR-Cas9-mediated genome editing for the correction of a TGFBI mutation in GCD patient-derived primary corneal keratocytes via homology-directed repair (HDR). To correct genetic defects in GCD patient cells, we designed a disease-specific guide RNA (gRNA) targeting the R124H mutation of TGFBI, which causes GCD type 2 (GCD2). An R124H mutation in primary human corneal keratocytes derived from a GCD2 patient was corrected by delivering a CRISPR plasmid expressing Cas9/gRNA and a single-stranded oligodeoxynucleotide HDR donor template in vitro. The gene correction efficiency was 20.6% in heterozygous cells and 41.3% in homozygous cells. No off-target effects were detected. These results reveal a new therapeutic strategy for GCD2; this method may also be applicable to other heredity corneal diseases

Effects of interactions between the constituents of chitosan-edible films on their physical properties

The main objective of this work was to evaluate the effect of chitosan and plasticizer concentrations and oil presence on the physical and mechanical properties of edible films. The effect of the film constituents and their in-between interactions were studied through the evaluation of permeability, opacity and mechanical properties. The effects of the studied variables (concentrations of chitosan, plasticizer and oil) were analysed according to a 2 3 factorial design. Pareto charts were used to identify the most significant factors in the studied properties (water vapour, oxygen and carbon dioxide permeability; opacity; tensile strength; elongation at break and Young's modulus). When addressing the influence of the interactions between the films' constituents on the properties above, results show that chitosan and plasticizer concentrations are the most significant factors affecting most of the studied properties, while oil incorporation has shown to be of a great importance in the particular case of transport properties (gas permeability), essentially due to its hydrophobicity. Water vapour permeability values (ranging from 1. 62 × 10 -11 to 4. 24 × 10 -11 g m -1 s -1 Pa -1) were half of those reported for cellophane films. Also the mechanical properties (tensile strength values from 0. 43 to 13. 72 MPa and elongation-at-break values from 58. 62% to 166. 70%) were in the range of those reported for LDPE and HDPE. Based on these results, we recommend the use of 1. 5% (w/w) chitosan concentration to produce films, where the oil and plasticizer proportions will have to be adjusted in a case-by-case basis according to the use intended for the material. This work provides a useful guide to the formulation of chitosan-based film-forming solutions for food packaging applications.The author MA Cerqueira is a recipient of a fellowship from Fundacao para a Ciencia e Tecnologia (FCT, SFRH/BD/23897/2005) and BWS Souza is a recipient of a fellowship from the Coordenacao Aperfeicoamento de Pessoal de Nivel Superior, Brazil (Capes, Brazil)

Are Better Workers Also Better Humans? On Pharmacological Cognitive Enhancement in the Workplace and Conflicting Societal Domains

The article investigates the sociocultural implications of the changing modern workplace and of pharmacological cognitive enhancement (PCE) as a potential adaptive tool from the viewpoint of social niche construction. We will attempt to elucidate some of the sociocultural and technological trends that drive and influence the characteristics of this specific niche, and especially to identify the kind of capabilities and adaptations that are being promoted, and to ascertain the capabilities and potentialities that might become diminished as a result. In this context, we will examine what PCE is, and how and why it might be desirable as a tool for adaptation within the workplace. As human beings are, or at least should be allowed to be, more than merely productive, able-bodied and able-minded workers, we will further examine how adaptation to the workplace niche could result in problems in other domains of modern societal life that require the same or other cognitive capabilities. In this context we will also focus on the concept of responsibility and how it pertains to PCE and the modern workplace niche. This will shed some light on the kind of trends related to workplace niche construction, PCE and capability promotion that we can expect in the future, and on the contexts in which this might be either beneficial or detrimental to the individual as a well-rounded human being, and to other members of society

Trust in Science: CRISPR-Cas9 and the Ban on Human Germline Editing

This is the final version of the article. Available from Springer Verlag via the DOI in this record.In 2015 scientists called for a partial ban on genome editing in human germline cells. This call was a response to the rapid development of the CRISPR-Cas9 system, a molecular tool that allows researchers to modify genomic DNA in living organisms with high precision and ease of use. Importantly, the ban was meant to be a trust-building exercise that promises a 'prudent' way forward. The goal of this paper is to analyse whether the ban can deliver on this promise. To do so the focus will be put on the precedent on which the current ban is modelled, namely the Asilomar ban on recombinant DNA technology. The analysis of this case will show (a) that the Asilomar ban was successful because of a specific two-step containment strategy it employed and (b) that this two-step approach is also key to making the current ban work. It will be argued, however, that the Asilomar strategy cannot be transferred to human genome editing and that the current ban therefore fails to deliver on its promise. The paper will close with a reflection on the reasons for this failure and on what can be learned from it about the regulation of novel molecular tools.The research leading to this paper has received funding from the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement No. 324186

CRISPR-Cas orthologues and variants: optimizing the repertoire, specificity and delivery of genome engineering tools

Robust and cost-effective genome editing in a diverse array of cells and model organisms is now possible thanks to the discovery of the RNA-guided endonucleases of the CRISPR-Cas system. The commonly used Cas9 of Streptococcus pyogenes shows high levels of activity but, depending on the application, has been associated with some shortcomings. Firstly, the enzyme has been shown to cause mutagenesis at genomic sequences resembling the target sequence. Secondly, the stringent requirement for a specific motif adjacent to the selected target site can limit the target range of this enzyme. Lastly, the physical size of Cas9 challenges the efficient delivery of genomic engineering tools based on this enzyme as viral particles for potential therapeutic applications. Related and parallel strategies have been employed to address these issues. Taking advantage of the wealth of structural information that is becoming available for CRISPR-Cas effector proteins, Cas9 has been redesigned by mutagenizing key residues contributing to activity and target recognition. The protein has also been shortened and redesigned into component subunits in an attempt to facilitate its efficient delivery. Furthermore, the CRISPR-Cas toolbox has been expanded by exploring the properties of Cas9 orthologues and other related effector proteins from diverse bacterial species, some of which exhibit different target site specificities and reduced molecular size. It is hoped that the improvements in accuracy, target range and efficiency of delivery will facilitate the therapeutic application of these site-specific nucleases

Applications of CRISPR–Cas systems in neuroscience

Genome-editing tools, and in particular those based on CRISPR-Cas (clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03

Measurement and interpretation of same-sign W boson pair production in association with two jets in pp collisions at s = 13 TeV with the ATLAS detector

This paper presents the measurement of fducial and diferential cross sections for both the inclusive and electroweak production of a same-sign W-boson pair in association with two jets (W±W±jj) using 139 fb−1 of proton-proton collision data recorded at a centre-of-mass energy of √s = 13 TeV by the ATLAS detector at the Large Hadron Collider. The analysis is performed by selecting two same-charge leptons, electron or muon, and at least two jets with large invariant mass and a large rapidity diference. The measured fducial cross sections for electroweak and inclusive W±W±jj production are 2.92 ± 0.22 (stat.) ± 0.19 (syst.)fb and 3.38±0.22 (stat.)±0.19 (syst.)fb, respectively, in agreement with Standard Model predictions. The measurements are used to constrain anomalous quartic gauge couplings by extracting 95% confdence level intervals on dimension-8 operators. A search for doubly charged Higgs bosons H±± that are produced in vector-boson fusion processes and decay into a same-sign W boson pair is performed. The largest deviation from the Standard Model occurs for an H±± mass near 450 GeV, with a global signifcance of 2.5 standard deviations