Gene syntax defines supercoiling-mediated transcriptional feedback.

Gene syntax-the order and arrangement of genes and their regulatory elements-shapes the dynamic coordination of both natural and synthetic gene circuits. Transcription at one locus profoundly impacts the transcription of nearby adjacent genes, but the molecular basis of this effect remains poorly understood. Here, using integrated reporter circuits in human cells, we show that supercoiling-mediated feedback regulates expression of adjacent genes in a syntax-specific manner.

STRAIGHT-IN Dual: a platform for dual, single-copy integrations of DNA payloads and gene circuits into human induced pluripotent stem cells.

Targeting DNA payloads into human (h)iPSCs involves multiple time-consuming, inefficient steps that must be repeated for each construct. Here, we present STRAIGHT-IN Dual, which enables simultaneous, allele-specific, single-copy integration of two DNA payloads with 100% efficiency within one week. Notably, STRAIGHT-IN Dual leverages the STRAIGHT-IN platform to allow near-scarless cargo integration, facilitating the recycling of components for subsequent cellular modifications.

Rewinding the Tape to Identify Intrinsic Determinants of Reprogramming Potential.

Via retrospective isolation of clones using Rewind, Jain et al. identified primed states of cells that reprogram to induced pluripotent stem cells. Examining clones, they find that cells retain memory of over several rounds of cell division.

Model-guided design of microRNA-based gene circuits supports precise dosage of transgenic cargoes into diverse primary cells.

To realize the potential of engineered cells in therapeutic applications, transgenes must be expressed within the window of therapeutic efficacy. Differences in copy number and other sources of extrinsic noise generate variance in transgene expression and limit the performance of synthetic gene circuits. In a therapeutic context, supraphysiological expression of transgenes can compromise engineered phenotypes and lead to toxicity.

Programmable promoter editing for precise control of transgene expression.

Subtle changes in gene expression direct cells to distinct cellular states. Identifying and controlling dose-dependent transgenes require tools for precisely titrating expression. To this end, we developed a highly modular, extensible framework called DIAL for building editable promoters that allow for fine-scale, heritable changes in transgene expression.

The dual orexin receptor antagonist suvorexant in alcohol use disorder and comorbid insomnia: A case report.

Key Clinical Message: This case suggests using dual orexin receptor antagonists to treat alcohol use disorder and comorbid sleep disorders may be effective, commencing treatment in withdrawal and continuing it to prevent relapse.

Abstract: Effective medications for the treatment of alcohol use disorder are limited. This is partially due to the heterogenous nature of the symptomatology associated with alcohol use disorder and the abundance of presenting comorbidities.

Accelerating Diverse Cell-Based Therapies Through Scalable Design.

Augmenting cells with novel, genetically encoded functions will support therapies that expand beyond natural capacity for immune surveillance and tissue regeneration. However, engineering cells at scale with transgenic cargoes remains a challenge in realizing the potential of cell-based therapies. In this review, we introduce a range of applications for engineering primary cells and stem cells for cell-based therapies.

Proliferation history and transcription factor levels drive direct conversion.

The sparse and stochastic nature of reprogramming has obscured our understanding of how transcription factors drive cells to new identities. To overcome this limit, we developed a compact, portable reprogramming system that increases direct conversion of fibroblasts to motor neurons by two orders of magnitude. We show that subpopulations with different reprogramming potentials are distinguishable by proliferation history.

RNA-based controllers for engineering gene and cell therapies.

Engineered RNA-based genetic controllers provide compact, tunable, post-transcriptional gene regulation. As RNA devices are generally small, these devices are portable to DNA and RNA viral vectors. RNA tools have recently expanded to allow reading and editing of endogenous RNAs for profiling and programming of transcriptional states.

The sound of silence: Transgene silencing in mammalian cell engineering.

To elucidate principles operating in native biological systems and to develop novel biotechnologies, synthetic biology aims to build and integrate synthetic gene circuits within native transcriptional networks. The utility of synthetic gene circuits for cell engineering relies on the ability to control the expression of all constituent transgene components. Transgene silencing, defined as the loss of expression over time, persists as an obstacle for engineering primary cells and stem cells with transgenic cargos.

Supercoiling-mediated feedback rapidly couples and tunes transcription.

Transcription induces a wave of DNA supercoiling, altering the binding affinity of RNA polymerases and reshaping the biochemical landscape of gene regulation. As supercoiling rapidly diffuses, transcription dynamically reshapes the regulation of proximal genes, forming a complex feedback loop. However, a theoretical framework is needed to integrate biophysical regulation with biochemical transcriptional regulation.

Evaluation of Lee et al.: Clarity and interpretation of mutual information in promoter transfer functions.

One snapshot of the peer review process for "Mapping the dynamic transfer functions of eukaryotic gene regulation" (Lee et al., 2021) appears below.

Single cell biology-a Keystone Symposia report.

Single cell biology has the potential to elucidate many critical biological processes and diseases, from development and regeneration to cancer. Single cell analyses are uncovering the molecular diversity of cells, revealing a clearer picture of the variation among and between different cell types. New techniques are beginning to unravel how differences in cell state-transcriptional, epigenetic, and other characteristics-can lead to different cell fates among genetically identical cells, which underlies complex processes such as embryonic development, drug resistance, response to injury, and cellular reprogramming.

Synthetic gene circuits as tools for drug discovery.

Within mammalian systems, there exists enormous opportunity to use synthetic gene circuits to enhance phenotype-based drug discovery, to map the molecular origins of disease, and to validate therapeutics in complex cellular systems. While drug discovery has relied on marker staining and high-content imaging in cell-based assays, synthetic gene circuits expand the potential for precision and speed. Here we present a vision of how circuits can improve the speed and accuracy of drug discovery by enhancing the efficiency of hit triage, capturing disease-relevant dynamics in cell-based assays, and simplifying validation and readouts from organoids and microphysiological systems (MPS).

Engineering cell fate: Applying synthetic biology to cellular reprogramming.

Cellular reprogramming drives cells from one stable identity to a new cell fate. By generating a diversity of previously inaccessible cell types from diverse genetic backgrounds, cellular reprogramming is rapidly transforming how we study disease. However, low efficiency and limited maturity have limited the adoption of -derived cellular models.

Understanding and Engineering Chromatin as a Dynamical System across Length and Timescales.

Connecting the molecular structure and function of chromatin across length and timescales remains a grand challenge to understanding and engineering cellular behaviors. Across five orders of magnitude, dynamic processes constantly reshape chromatin structures, driving spaciotemporal patterns of gene expression and cell fate. Through the interplay of structure and function, the genome operates as a highly dynamic feedback control system.

Mitigating Antagonism between Transcription and Proliferation Allows Near-Deterministic Cellular Reprogramming.

Although cellular reprogramming enables the generation of new cell types for disease modeling and regenerative therapies, reprogramming remains a rare cellular event. By examining reprogramming of fibroblasts into motor neurons and multiple other somatic lineages, we find that epigenetic barriers to conversion can be overcome by endowing cells with the ability to mitigate an inherent antagonism between transcription and DNA replication. We show that transcription factor overexpression induces unusually high rates of transcription and that sustaining hypertranscription and transgene expression in hyperproliferative cells early in reprogramming is critical for successful lineage conversion.

Comparative genomic analysis of embryonic, lineage-converted and stem cell-derived motor neurons.

Advances in stem cell science allow the production of different cell types either through the recapitulation of developmental processes, often termed 'directed differentiation', or the forced expression of lineage-specific transcription factors. Although cells produced by both approaches are increasingly used in translational applications, their quantitative similarity to their primary counterparts remains largely unresolved. To investigate the similarity between -derived and primary cell types, we harvested and purified mouse spinal motor neurons and compared them with motor neurons produced by transcription factor-mediated lineage conversion of fibroblasts or directed differentiation of pluripotent stem cells.

Feedback loops in biological networks.

We introduce fundamental concepts for the design of dynamics and feedback in molecular networks modeled with ordinary differential equations. We use several examples, focusing in particular on the mitogen-activated protein kinase (MAPK) pathway, to illustrate the concept that feedback loops are fundamental in determining the overall dynamic behavior of a system. Often, these loops have a structural function and unequivocally define the system behavior.

Dynamically reshaping signaling networks to program cell fate via genetic controllers.

Engineering of cell fate through synthetic gene circuits requires methods to precisely implement control around native decision-making pathways and offers the potential to direct cell processes. We demonstrate a class of genetic control systems, molecular network diverters, that interface with a native signaling pathway to route cells to divergent fates in response to environmental signals without modification of native genetic material. A method for identifying control points within natural networks is described that enables the construction of synthetic control systems that activate or attenuate native pathways to direct cell fate.

Improving spinal trauma management in non-specialist centres.

Fractures of the vertebral column are increasing in incidence. Even though spinal trauma is increasingly being managed in specialist units, these patients often still initially present to district general hospitals. Due to lack of exposure to these patients, the attending Orthopaedic Senior House Officer may not always be aware of current best practice in the acute management of these patients beyond immediate Advance Trauma Life Support measures.