The historical reliance of biological research on the use of animal models has sometimes made it challenging to address questions that are specific to the understanding of human biology and disease. ...But with the advent of human organoids - which are stem cell-derived 3D culture systems - it is now possible to re-create the architecture and physiology of human organs in remarkable detail. Human organoids provide unique opportunities for the study of human disease and complement animal models. Human organoids have been used to study infectious diseases, genetic disorders and cancers through the genetic engineering of human stem cells, as well as directly when organoids are generated from patient biopsy samples. This Review discusses the applications, advantages and disadvantages of human organoids as models of development and disease and outlines the challenges that have to be overcome for organoids to be able to substantially reduce the need for animal experiments.
CRISPR-Cas9-based genetic screens are a powerful new tool in biology. By simply altering the sequence of the single-guide RNA (sgRNA), one can reprogram Cas9 to target different sites in the genome ...with relative ease, but the on-target activity and off-target effects of individual sgRNAs can vary widely. Here, we use recently devised sgRNA design rules to create human and mouse genome-wide libraries, perform positive and negative selection screens and observe that the use of these rules produced improved results. Additionally, we profile the off-target activity of thousands of sgRNAs and develop a metric to predict off-target sites. We incorporate these findings from large-scale, empirical data to improve our computational design rules and create optimized sgRNA libraries that maximize on-target activity and minimize off-target effects to enable more effective and efficient genetic screens and genome engineering.
Cerebral deposition of abnormally aggregated alpha-synuclein (alphaSyn) is a neuropathological hallmark of Parkinson's disease (PD). PD-associated alphaSyn (alphaSyn.sup.PD) aggregates can act as ...proteinaceous nuclei ("seeds") able of self-templated propagation. Since this is strikingly reminiscent to properties of proteinaceous infectious particles (prions), lessons learned from prion diseases suggest to test whether transferred alphaSyn.sup.PD can propagate and induce neurological impairments or disease in a new host. Two studies that addressed this question provided divergent results. Intracerebral (i.c.) injection of Lewy body extracts from PD patients caused cerebral alphaSyn pathology, as well as nigrostriatal neurodegeneration, of wild-type mice and macaques, with the mice also showing motor impairments (Recasens et al. 2014, Ann Neurol 75:351-362). In contrast, i.c. transmission of homogenates from PD brains did not stimulate, after "> 360" days post-injection (dpi), pathological alphaSyn conversion or clinical symptoms in transgenic TgM83.sup.+/- mice hemizygously expressing mutated (A53T) human alphaSyn (Prusiner et al. 2015, PNAS 112:E5308-E5317). To advance the assessment of possible alphaSyn.sup.PD hazards by providing further data, we examined neuropathological and clinical effects upon i.c. transmission of brain, stomach wall and muscle tissue as well as blood from PD patients in TgM83.sup.+/- mice up to 612 dpi. This revealed a subtle, yet distinctive stimulation of localized alphaSyn aggregation in the somatodendritic compartment and dystrophic neurites of individual or focally clustered cerebral neurons after challenge with brain and stomach wall homogenates. No such effect was observed with transmitted blood or homogenized muscle tissue. The detected stimulation of alphaSyn aggregation was not accompanied by apparent motor impairments or overt neurological disease in TgM83.sup.+/- mice. Our study substantiated that transmitted alphaSyn.sup.PD seeds, including those from the stomach wall, are able to propagate in new mammalian hosts. The consequences of such propagation and potential safeguards need to be further investigated.
Recent studies into the global causes of severe diarrhoea in young children have identified the protozoan parasite Cryptosporidium as the second most important diarrhoeal pathogen after rotavirus. ...Diarrhoeal disease is estimated to be responsible for 10.5% of overall child mortality. Cryptosporidium is also an opportunistic pathogen in the contexts of human immunodeficiency virus (HIV)-caused AIDS and organ transplantation. There is no vaccine and only a single approved drug that provides no benefit for those in gravest danger: malnourished children and immunocompromised patients. Cryptosporidiosis drug and vaccine development is limited by the poor tractability of the parasite, which includes a lack of systems for continuous culture, facile animal models, and molecular genetic tools. Here we describe an experimental framework to genetically modify this important human pathogen. We established and optimized transfection of C. parvum sporozoites in tissue culture. To isolate stable transgenics we developed a mouse model that delivers sporozoites directly into the intestine, a Cryptosporidium clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, and in vivo selection for aminoglycoside resistance. We derived reporter parasites suitable for in vitro and in vivo drug screening, and we evaluated the basis of drug susceptibility by gene knockout. We anticipate that the ability to genetically engineer this parasite will be transformative for Cryptosporidium research. Genetic reporters will provide quantitative correlates for disease, cure and protection, and the role of parasite genes in these processes is now open to rigorous investigation.
Synthetic Biology Open Language (SBOL) Visual is a graphical standard for genetic engineering. It consists of symbols representing DNA subsequences, including regulatory elements and DNA assembly ...features. These symbols can be used to draw illustrations for communication and instruction, and as image assets for computer-aided design. SBOL Visual is a community standard, freely available for personal, academic, and commercial use (Creative Commons CC0 license). We provide prototypical symbol images that have been used in scientific publications and software tools. We encourage users to use and modify them freely, and to join the SBOL Visual community: http://www.sbolstandard.org/visual.
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![Transmissible [alpha]-synuc...](http://api.summon.serialssolutions.com/2.0.0/image/issn/XR4PS5YY4K/0001-6322/small "Transmissible [alpha]-synuclein seeding activity in brain and stomach of patients with Parkinson's disease")
