Engineering multiple levels of specificity in an RNA viral vector

Synthetic molecular circuits could provide powerful therapeutic capabilities, but delivering them to specific cell types and controlling them remains challenging. An ideal "smart" viral delivery system would enable controlled release of viral vectors from "sender" cells, conditional entry into target cells based on cell-surface proteins, conditional replication specifically in target cells based on their intracellular protein content, and an evolutionarily robust system that allows viral elimination with drugs. Here, combining diverse technologies and components, including pseudotyping, engineered bridge proteins, degrons, and proteases, we demonstrate each of these control modes in a model system based on the rabies virus. This work shows how viral and protein engineering can enable delivery systems with multiple levels of control to maximize therapeutic specificity

Programmable protein circuits in living cells

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications

Programmable protein circuits in living cells

Synthetic protein-level circuits could enable engineering of powerful new cellular behaviors. Rational protein circuit design would be facilitated by a composable protein-protein regulation system in which individual protein components can regulate one another to create a variety of different circuit architectures. In this study, we show that engineered viral proteases can function as composable protein components, which can together implement a broad variety of circuit-level functions in mammalian cells. In this system, termed CHOMP (circuits of hacked orthogonal modular proteases), input proteases dock with and cleave target proteases to inhibit their function. These components can be connected to generate regulatory cascades, binary logic gates, and dynamic analog signal-processing functions. To demonstrate the utility of this system, we rationally designed a circuit that induces cell death in response to upstream activators of the Ras oncogene. Because CHOMP circuits can perform complex functions yet be encoded as single transcripts and delivered without genomic integration, they offer a scalable platform to facilitate protein circuit engineering for biotechnological applications

Topological Organization of Ventral Tegmental Area Connectivity Revealed by Viral-Genetic Dissection of Input-Output Relations

Viral-genetic tracing techniques have enabled mesoscale mapping of neuronal connectivity by teasing apart inputs to defined neuronal populations in regions with heterogeneous cell types. We previously observed input biases to output-defined ventral tegmental area dopamine (VTA-DA) neurons. Here, we further dissect connectivity in the VTA by defining input-output relations of neurochemically and output-defined neuronal populations. By expanding our analysis to include input patterns to subtypes of excitatory (vGluT2-expressing) or inhibitory (GAD2-expressing) populations, we find that the output site, rather than neurochemical phenotype, correlates with whole-brain inputs of each subpopulation. Lastly, we find that biases in input maps to different VTA neurons can be generated using publicly available whole-brain output mapping datasets. Our comprehensive dataset and detailed spatial analysis suggest that connection specificity in the VTA is largely a function of the spatial location of the cells within the VTA

Cas9-triggered chain ablation of cas9 as a gene drive brake

With the advent of clustered, regularly interspaced, short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) technology, researchers can construct gene drives that can bias the inheritance of edited alleles to alter entire populations. As demonstrated with the mutagenic chain reaction in Drosophila4, the CRISPR-Cas9 system can propagate genomic modification together with the genome-editing machinery itself. Although gene drives might have the potential to control insect-borne diseases and agricultural pests, substantial concerns have been raised over unanticipated ecological consequences as a result of drive use. Here we report the development of a potential Cas9-based gene drive 'brake' that remains inert in a wild-type genome but is activated by Cas9 to both cleave the genomic cas9 sequence and to convert an incoming cas9 allele into a brake. This means that the propagation of the brake is favored in a cas9-carrying population

Regional glutamine deficiency in tumours promotes dedifferentiation through inhibition of histone demethylation

Poorly organized tumour vasculature often results in areas of limited nutrient supply and hypoxia. Despite our understanding of solid tumour responses to hypoxia, how nutrient deprivation regionally affects tumour growth and therapeutic response is poorly understood. Here, we show that the core region of solid tumours displayed glutamine deficiency compared with other amino acids. Low glutamine in tumour core regions led to dramatic histone hypermethylation due to decreased α-ketoglutarate levels, a key cofactor for the Jumonji-domain-containing histone demethylases. Using patient-derived ^(V600E)BRAF melanoma cells, we found that low-glutamine-induced histone hypermethylation resulted in cancer cell dedifferentiation and resistance to BRAF inhibitor treatment, which was largely mediated by methylation on H3K27, as knockdown of the H3K27-specific demethylase KDM6B and the methyltransferase EZH2 respectively reproduced and attenuated the low-glutamine effects in vitro and in vivo. Thus, intratumoral regional variation in the nutritional microenvironment contributes to tumour heterogeneity and therapeutic response

Synthetic biology: Precision timing in a cell

A 16-year-old synthetic genetic circuit that produces gene-expression oscillations in bacterial cells has been given an upgrade, making it an exceptionally precise biological clock

Extremely sparse olfactory inputs are sufficient to mediate innate aversion in Drosophila.

Innate attraction and aversion to odorants are observed throughout the animal kingdom, but how olfactory circuits encode such valences is not well understood, despite extensive anatomical and functional knowledge. In Drosophila melanogaster, ~50 types of olfactory receptor neurons (ORNs) each express a unique receptor gene, and relay information to a cognate type of projection neurons (PNs). To examine the extent to which the population activity of ORNs is required for olfactory behavior, we developed a genetic strategy to block all ORN outputs, and then to restore output in specific types. Unlike attraction, aversion was unaffected by simultaneous silencing of many ORNs, and even single ORN types previously shown to convey neutral valence sufficed to mediate aversion. Thus, aversion may rely on specific activity patterns in individual ORNs rather than the number or identity of activated ORNs. ORN activity is relayed into the brain by downstream circuits, with excitatory PNs (ePN) representing a major output. We found that silencing the majority of ePNs did not affect aversion, even when ePNs directly downstream of single restored ORN types were silenced. Our data demonstrate the robustness of olfactory aversion, and suggest that its circuit mechanism is qualitatively different from attraction