Showing papers by "Ron Weiss published in 2014"

Realizing the potential of synthetic biology

Abstract: Synthetic biology, despite still being in its infancy, is increasingly providing valuable information for applications in the clinic, the biotechnology industry and in basic molecular research. Both its unique potential and the challenges it presents have brought together the expertise of an eclectic group of scientists, from cell biologists to engineers. In this Viewpoint article, five experts discuss their views on the future of synthetic biology, on its main achievements in basic and applied science, and on the bioethical issues that are associated with the design of new biological systems.

Highly-efficient Cas9-mediated transcriptional programming

Abstract: The RNA-guided bacterial nuclease Cas9 can be reengineered as a programmable transcription factor by a series of changes to the Cas9 protein in addition to the fusion of a transcriptional activation domain (AD). However, the modest levels of gene activation achieved by current Cas9 activators have limited their potential applications. Here we describe the development of an improved transcriptional regulator through the rational design of a tripartite activator, VP64-p65-Rta (VPR), fused to Cas9. We demonstrate its utility in activating expression of endogenous coding and non-coding genes, targeting several genes simultaneously and stimulating neuronal differentiation of induced pluripotent stem cells (iPSCs).

CRISPR transcriptional repression devices and layered circuits in mammalian cells

Abstract: A key obstacle to creating sophisticated genetic circuits has been the lack of scalable device libraries. Here we present a modular transcriptional repression architecture based on clustered regularly interspaced palindromic repeats (CRISPR) system and examine approaches for regulated expression of guide RNAs in human cells. Subsequently we demonstrate that CRISPR regulatory devices can be layered to create functional cascaded circuits, which provide a valuable toolbox for engineering purposes.

Rapid neurogenesis through transcriptional activation in human stem cells

Abstract: Advances in cellular reprogramming and stem cell differentiation now enable ex vivo studies of human neuronal differentiation. However, it remains challenging to elucidate the underlying regulatory programs because differentiation protocols are laborious and often result in low neuron yields. Here, we overexpressed two Neurogenin transcription factors in human-induced pluripotent stem cells and obtained neurons with bipolar morphology in 4 days, at greater than 90% purity. The high purity enabled mRNA and microRNA expression profiling during neurogenesis, thus revealing the genetic programs involved in the rapid transition from stem cell to neuron. The resulting cells exhibited transcriptional, morphological and functional signatures of differentiated neurons, with greatest transcriptional similarity to prenatal human brain samples. Our analysis revealed a network of key transcription factors and microRNAs that promoted loss of pluripotency and rapid neurogenesis via progenitor states. Perturbations of key transcription factors affected homogeneity and phenotypic properties of the resulting neurons, suggesting that a systems-level view of the molecular biology of differentiation may guide subsequent manipulation of human stem cells to rapidly obtain diverse neuronal types.

A load driver device for engineering modularity in biological networks

Abstract: The behavior of gene modules in complex synthetic circuits is often unpredictable. After joining modules to create a circuit, downstream elements (such as binding sites for a regulatory protein) apply a load to upstream modules that can negatively affect circuit function. Here we devised a genetic device named a load driver that mitigates the impact of load on circuit function, and we demonstrate its behavior in Saccharomyces cerevisiae. The load driver implements the design principle of timescale separation: inclusion of the load driver's fast phosphotransfer processes restores the capability of a slower transcriptional circuit to respond to time-varying input signals even in the presence of substantial load. Without the load driver, we observed circuit behavior that suffered from a 76% delay in response time and a 25% decrease in system bandwidth due to load. With the addition of a load driver, circuit performance was almost completely restored. Load drivers will serve as fundamental building blocks in the creation of complex, higher-level genetic circuits.

A platform for rapid prototyping of synthetic gene networks in mammalian cells

Abstract: Mammalian synthetic biology may provide novel therapeutic strategies, help decipher new paths for drug discovery and facilitate synthesis of valuable molecules. Yet, our capacity to genetically program cells is currently hampered by the lack of efficient approaches to streamline the design, construction and screening of synthetic gene networks. To address this problem, here we present a framework for modular and combinatorial assembly of functional (multi)gene expression vectors and their efficient and specific targeted integration into a welldefined chromosomal context in mammalian cells. We demonstrate the potential of this framework by assemblingandintegratingdifferentfunctionalmammalian regulatory networks including the largest gene circuit built and chromosomally integrated to date (6 transcription units, 27kb) encoding an inducible memory device. Using a library of 18 different circuits as a proof of concept, we also demonstrate that our method enables one-pot/single-flask chromosomal integration and screening of circuit libraries. This rapid and powerful prototyping platform is well suited for comparative studies of genetic regulatory elements, genes and multi-gene circuits as well as facile development of libraries of isogenic engineered cell lines.

Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells

Abstract: Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repres...

Systematic transfer of prokaryotic sensors and circuits to mammalian cells.

Abstract: Prokaryotic regulatory proteins respond to diverse signals and represent a rich resource for building synthetic sensors and circuits. The TetR family contains >105 members that use a simple mechanism to respond to stimuli and bind distinct DNA operators. We present a platform that enables the transfer of these regulators to mammalian cells, which is demonstrated using human embryonic kidney (HEK293) and Chinese hamster ovary (CHO) cells. The repressors are modified to include nuclear localization signals (NLS) and responsive promoters are built by incorporating multiple operators. Activators are also constructed by modifying the protein to include a VP16 domain. Together, this approach yields 15 new regulators that demonstrate 19- to 551-fold induction and retain both the low levels of crosstalk in DNA binding specificity observed between the parent regulators in Escherichia coli, as well as their dynamic range of activity. By taking advantage of the DAPG small molecule sensing mediated by the PhlF repres...

Sensors for micro bio robots via synthetic biology

Abstract: Microscale robots offer an unprecedented opportunity to perform tasks at resolutions approaching 1 μm, but the great majority of research to this point focuses on actuation and control. Potential applications for microrobots can be considerably expanded by integrating sensing, signal processing and feedback into the system. In this work, we demonstrate that technologies from the field of synthetic biology may be directly integrated into microrobotic systems to create cell-based programmable mobile sensors, with signal processors and memory units. Specifically, we integrate genetically engineered, ultraviolet light-sensing bacteria with magnetic microrobots, creating the first controllable biological microrobot that is capable of exploring, recording and reporting on the state of the microscale environment. We demonstrate two proof-of-concept prototypes: (a) an integrated microrobot platform that is able to sense biochemical signals, and (b) a microrobot platform that is able to deploy biosensor payloads to monitor biochemical signals, both in a biological environment. These results have important implications for integrated micro-bio-robotic systems for applications in biological engineering and research.

RNAi Synthetic Logic Circuits for Sensing, Information Processing, and Actuation

Abstract: Noncoding small RNAs regulate gene expression through complex RNA interference (RNAi) signaling networks. The elucidation of molecular mechanisms underlying RNAi and the discovery of commonly occurring transcriptional and post-transcriptional motifs have enabled the construction of RNAi-based sensors and devices used for engineering genetic modules and logic circuits that offer sophisticated control of biological systems. In this chapter, recent progress in the design and implementation of RNAi-based logic circuits for the sensing and processing of multiple molecular signals to generate programmed biological actuation is discussed. Keywords: RNA interference; Logic circuit; Genetic circuit

Engineering a heterogeneous tissue from pluripotent stem cells

Abstract: Aspects of the present disclosure are directed to methods and compositions for the production of heterogeneous tissue from human induced pluripotent stem (hiPS) cells.

Affinity Weighted Embedding

Abstract: Supervised linear embedding models like WSABIE (Weston et al., 2011) and supervised semantic indexing (Bai et al., 2010) have proven successful at ranking, recommendation and annotation tasks. However, despite being scalable to large datasets they do not take full advantage of the extra data due to their linear nature, and we believe they typically underfit. We propose a new class of models which aim to provide improved performance while retaining many of the benefits of the existing class of embedding models. Our approach works by reweighting each component of the embedding of features and labels with a potentially nonlinear affinity function. We describe several variants of the family, and show its usefulness on several datasets.