Simulating a in the human genome: Design and evaluation of recombinase genetic programs that count to three.

Pluripotent cells specialize into numerous cell types by receiving external signals, making fate decisions, and executing differentiation functions - a paradigm similar to computer algorithms. While advances in biosensor design have enabled cells to respond to diverse stimuli, the ability to maintain a synthetic memory of the cell's experiences that then informs its behaviors remains elusive. Here, we developed a system for cellular memory-driven behaviors by simulating a "for-loop" that counts to three using recombinase STepwise gene Expression Programs (STEPs).

Iterative transcription factor screening enables rapid generation of microglia-like cells from human iPSC.

Differentiation of induced pluripotent stem cells (iPSCs) into specialized cell types is essential for uncovering cell-type specific molecular mechanisms and interrogating cellular function. Transcription factor screens have enabled efficient production of a few cell types; however, engineering cell types that require complex transcription factor combinations remains challenging. Here, we report an iterative, high-throughput single-cell transcription factor screening method that enables the identification of transcription factor combinations for specialized cell differentiation, which we validated by differentiating human microglia-like cells.

Machine-guided cell-fate engineering.

The creation of induced pluripotent stem cells (iPSCs) has enabled scientists to explore the function, mechanisms, and differentiation processes of many types of cells. One of the fastest and most efficient approaches is transcription factor (TF) over-expression. However, finding the right combination of TFs to over-express to differentiate iPSCs directly into other cell types is a difficult task.

Algorithms for Autonomous Formation of Multicellular Shapes from Single Cells.

Multicellular organisms originate from a single cell, ultimately giving rise to mature organisms of heterogeneous cell type composition in complex structures. Recent work in the areas of stem cell biology and tissue engineering has laid major groundwork in the ability to convert certain types of cells into other types, but there has been limited progress in the ability to control the morphology of cellular masses as they grow. Contemporary approaches to this problem have included the use of artificial scaffolds, 3D bioprinting, and complex media formulations; however, there are no existing approaches to controlling this process purely through genetics and from a single-cell starting point.

helixCAM: A platform for programmable cellular assembly in bacteria and human cells.

Interactions between cells are indispensable for signaling and creating structure. The ability to direct precise cell-cell interactions would be powerful for engineering tissues, understanding signaling pathways, and directing immune cell targeting. In humans, intercellular interactions are mediated by cell adhesion molecules (CAMs).

Targeted intracellular delivery of Cas13 and Cas9 nucleases using bacterial toxin-based platforms.

Targeted delivery of therapeutic proteins toward specific cells and across cell membranes remains major challenges. Here, we develop protein-based delivery systems utilizing detoxified single-chain bacterial toxins such as diphtheria toxin (DT) and botulinum neurotoxin (BoNT)-like toxin, BoNT/X, as carriers. The system can deliver large protein cargoes including Cas13a, CasRx, Cas9, and Cre recombinase into cells in a receptor-dependent manner, although delivery of ribonucleoproteins containing guide RNAs is not successful.

Cell therapy strategies for COVID-19: Current approaches and potential applications.

Coronavirus disease 2019 (COVID-19) continues to burden society worldwide. Despite most patients having a mild course, severe presentations have limited treatment options. COVID-19 manifestations extend beyond the lungs and may affect the cardiovascular, nervous, and other organ systems.

Synthetic auxotrophy remains stable after continuous evolution and in coculture with mammalian cells.

Understanding the evolutionary stability and possible context dependence of biological containment techniques is critical as engineered microbes are increasingly under consideration for applications beyond biomanufacturing. While synthetic auxotrophy previously prevented from exhibiting detectable escape from batch cultures, its long-term effectiveness is unknown. Here, we report automated continuous evolution of a synthetic auxotroph while supplying a decreasing concentration of essential biphenylalanine (BipA).

A computer-guided design tool to increase the efficiency of cellular conversions.

Human cell conversion technology has become an important tool for devising new cell transplantation therapies, generating disease models and testing gene therapies. However, while transcription factor over-expression-based methods have shown great promise in generating cell types in vitro, they often endure low conversion efficiency. In this context, great effort has been devoted to increasing the efficiency of current protocols and the development of computational approaches can be of great help in this endeavor.

Algorithms for the selection of fluorescent reporters.

Molecular biologists rely on the use of fluorescent probes to take measurements of their model systems. These fluorophores fall into various classes (e.g.

A comprehensive library of human transcription factors for cell fate engineering.

Human pluripotent stem cells (hPSCs) offer an unprecedented opportunity to model diverse cell types and tissues. To enable systematic exploration of the programming landscape mediated by transcription factors (TFs), we present the Human TFome, a comprehensive library containing 1,564 TF genes and 1,732 TF splice isoforms. By screening the library in three hPSC lines, we discovered 290 TFs, including 241 that were previously unreported, that induce differentiation in 4 days without alteration of external soluble or biomechanical cues.

Building biosecurity for synthetic biology.

The fast-paced field of synthetic biology is fundamentally changing the global biosecurity framework. Current biosecurity regulations and strategies are based on previous governance paradigms for pathogen-oriented security, recombinant DNA research, and broader concerns related to genetically modified organisms (GMOs). Many scholarly discussions and biosecurity practitioners are therefore concerned that synthetic biology outpaces established biosafety and biosecurity measures to prevent deliberate and malicious or inadvertent and accidental misuse of synthetic biology's processes or products.

Needs and opportunities in bio-design automation: four areas for focus.

Bio-design automation (BDA) is an emerging field focused on computer-aided design, engineering principles, and automated manufacturing of biological systems. Here we discuss some outstanding challenges for bio-design that can be addressed by developing new tools for combinatorial engineering, equipment interfacing, next-generation sequencing, and workflow integration. These four areas, while not an exhaustive list of those that need to be addressed, could yield advances in bio-design, laboratory automation, and biometrology.

Design Automation in Synthetic Biology.

Design automation refers to a category of software tools for designing systems that work together in a workflow for designing, building, testing, and analyzing systems with a target behavior. In synthetic biology, these tools are called bio-design automation (BDA) tools. In this review, we discuss the BDA tools areas-specify, design, build, test, and learn-and introduce the existing software tools designed to solve problems in these areas.

Designing safety policies to meet evolving needs: iGEM as a testbed for proactive and adaptive risk management.

iGEM has spent the past decade encouraging teams to push their projects to the frontiers of synthetic biology. However, as project complexity increases, so too does the level of assumed risk. In the absence of a coherent international framework for evaluating these risks in synthetic biology, iGEM has recently engaged with the MIT Program on Emerging Technologies to develop a progressive approach for handling questions of safety and security.

Owl: electronic datasheet generator.

Owl ( www.owlcad.org ) is a biodesign automation tool that generates electronic datasheets for synthetic biological parts using common formatting.

Interactive assembly algorithms for molecular cloning.

Molecular biologists routinely clone genetic constructs from DNA segments and formulate plans to assemble them. However, manual assembly planning is complex, error prone and not scalable. We address this problem with an algorithm-driven DNA assembly planning software tool suite called Raven (http://www.

An end-to-end workflow for engineering of biological networks from high-level specifications.

We present a workflow for the design and production of biological networks from high-level program specifications. The workflow is based on a sequence of intermediate models that incrementally translate high-level specifications into DNA samples that implement them. We identify algorithms for translating between adjacent models and implement them as a set of software tools, organized into a four-stage toolchain: Specification, Compilation, Part Assignment, and Assembly.