SynBioFromLeukipposInstitute

Building smarter synthetic biological circuits
Synthetic genetic and biological regulatory circuits can enable logic functions to form the basis of biological computing; synthetic biology can also be used to control cell behaviors (see the Perspective by Glass and Alon). Andrews et al. used mathematical models and computer algorithms to combine standardized components and build programmable genetic sequential logic circuits. Such circuits can perform regulatory functions much like the biological checkpoint circuits of living cells. Circuits composed of interacting proteins could be used to bypass gene regulation, interfacing directly with cellular pathways without genome modification. Gao et al. engineered proteases that regulate one another, respond to diverse inputs that include oncogene activation, process signals, and conditionally activate responses such as those leading to cell death. This platform should facilitate development of “smart” therapeutic circuits for future biomedical applications.

An electrochemical biosensor based on Cu2+-dependent cleavage DNAzyme (cDNAzyme), Exponential Amplification Reaction (EXPAR), and single-strand triggered DNA hybridization chain reaction (HCR) was developed for detecting the copper (II) ions. This method capitalizes on the specific recognition of cDNAzyme, the single strand accumulated isothermal amplification of EXPAR, and the enzyme-free isothermal DNA assembly of HCR. In the presence of Cu2+, the catalytic chain of cDNAzyme split the substrate chain to produce single-stranded DNA (Oligo X). With the help of the EXPAR, the trace Oligo X was amplified and converted to the initiator DNA (Oligo Y). After adding the initiator DNA (Oligo Y) into nanochannel, massive DNA superstructures grew from the capture probe (CP) that were pre-immobilized on the nanochannel wall, resulting in a sharp decrease of the effective diameter of the nanochannel. As a result, the transmembrane ionic current significantly decreased due to the effective closing of the nanochannel. Finally, the copper (II) ion was detected by monitoring the changes of transmembrane ionic current. The developed electrochemical biosensor also displayed high selectivity for Cu2+, which could be useful for monitoring Cu2+ above ten picomole.

Engineering biological systems for the production of biofuels and bioproducts holds great potential to transform the bioeconomy, but often requires laborious, time-consuming design-build-test cycles. For decades cell-free systems have offered quick and facile approaches to study enzymes with hopes of informing cellular processes, mainly in the form of purified single-enzyme activity assays. Over the past 20 years, cell-free systems have grown to include multienzymatic systems, both purified and crude. By decoupling cellular growth objectives from enzyme pathway engineering objectives, cell-free systems provide a controllable environment to direct substrates toward a single, desired product. Cell-free approaches are being developed for prototyping and for biomanufacturing. In prototyping applications, the idea is to use cell-free systems to test and optimize biosynthetic pathways before implementation in live cells and scale-up. We present a detailed method for the generation of crude lysates for cell-free pathway prototyping, mix-and-match cell-free metabolic engineering using preenriched lysates, and cell-free protein synthesis driven cell-free metabolic engineering. The cell-free synthetic biology methods described herein are generalizable to any biosynthetic pathway of interest and provide a powerful approach to building pathways in crude lysates for the purpose of prototyping. The foundational principle of the presented approach is that we can construct discrete metabolic pathways through modular assembly of cell-free lysates containing enzyme components produced by overexpression in the lysate chassis strain or by cell-free protein synthesis (in vitro production). Overall, the modular and cell-free nature of our pathway prototyping framework is poised to facilitate multiplexed, automated study of biosynthetic pathways to inform systems-level cellular design.

The diversity, modularity, and efficacy of CRISPR-Cas systems are driving a biotechnological revolution. RNA-guided Cas enzymes have been adopted as tools to manipulate the genomes of cultured cells, animals, and plants, accelerating the pace of fundamental research and enabling clinical and agricultural breakthroughs. We describe the basic mechanisms that set the CRISPR-Cas toolkit apart from other programmable gene-editing technologies, highlighting the diverse and naturally evolved systems now functionalized as biotechnologies. We discuss the rapidly evolving landscape of CRISPR-Cas applications, from gene editing to transcriptional regulation, imaging, and diagnostics. Continuing functional dissection and an expanding landscape of applications position CRISPR-Cas tools at the cutting edge of nucleic acid manipulation that is rewriting biology.

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.

There is a need for large-scale, longitudinal studies to determine the mechanisms by which the gut microbiome and its interactions with the host affect human health and disease. Current methods for profiling the microbiome typically utilize next-generation sequencing applications that are expensive, slow, and complex. Here, we present a synthetic biology platform for affordable, on-demand, and simple analysis of microbiome samples using RNA toehold switch sensors in paper-based, cell-free reactions. We demonstrate species-specific detection of mRNAs from 10 different bacteria that affect human health and four clinically relevant host biomarkers. We develop a method to quantify mRNA using our toehold sensors and validate our platform on clinical stool samples by comparison to RT-qPCR. We further highlight the potential clinical utility of the platform by showing that it can be used to rapidly and inexpensively detect toxin mRNA in the diagnosis of Clostridium difficile infections.

Measuring biological data across time and space is critical for understanding complex biological processes and for various biosurveillance applications. However, such data are often inaccessible or difficult to directly obtain. Less invasive, more robust and higher-throughput biological recording tools are needed to profile cells and their environments. DNA-based cellular recording is an emerging and powerful framework for tracking intracellular and extracellular biological events over time across living cells and populations. Here, we review and assess DNA recorders that utilize CRISPR nucleases, integrases and base-editing strategies, as well as recombinase and polymerase-based methods. Quantitative characterization, modelling and evaluation of these DNA-recording modalities can guide their design and implementation for specific application areas.

Organic-inorganic hybrid nanoflowers (HNFs) are a class of flower-like hybrid materials self-assembled from metal ions and organic components, such as enzymes, antibodies, DNA and amino acids et al. Based on their properties of enhanced enzyme activity, stability, facile synthesis and excellent biocompatibility, HNFs enable them to be a highly versatile platform for latent applications in many realms, such as biological sensing, biomimetic catalyst, dye decolorization and support nanomaterials, etc. Compared with free enzymes, HNFs are potentially advantageous for biological applications owing to their enhanced catalytic ability and powerful load capacity. Herein, an overview of recent developments achieved in HNFs biosensing, including electrochemical biosensors, colorimetric biosensors, and point-of-care diagnostic devices, is provided. We summarized the preparation of different HNFs based on the type of metal ions and biomolecules used. The signal tags involved in the all-in-one hybrid nanoflowers are particularly emphasized. The challenges, future trends, and prospects associated with HNFs and their related materials for biosensing are also discussed.

A powerful gene-editing technique can stimulate dogs’ production of an important muscle protein, a finding that takes researchers a step closer to trying the technology in humans who have a fatal deficit in the same molecule.

People with the genetic disease Duchenne muscular dystrophy cannot synthesize dystrophin, a component of the scaffolding inside muscle cells. This deficiency results in muscle wasting and early death. The CRISPR–Cas9 gene-editing system has previously been used to trigger dystrophin production in mice, but Eric Olson at the University of Texas Southwestern Medical Center in Dallas and his colleagues sought to test the technique in a larger animal.

The researchers treated four dogs — all lacking dystrophin because of a genetic mutation — with the CRISPR–Cas9 system, which snipped out a short stretch of the animals’ DNA. This allowed the dogs’ cells to make dystrophin. Levels of the protein in one dog’s heart muscle reached 92% of normal.

Glucose and xylose are the two most abundant sugars in renewable lignocellulose sources; however, typically they cannot be simultaneously utilized due to carbon catabolite repression. N-acetylglucosamine (GlcNAc) is a typical nutraceutical and has many applications in the field of healthcare. Here, we have developed a gene repressor system based on xylose-induced CRISPR interference (CRISPRi) in Bacillus subtilis, aimed at downregulating the expression of three genes (zwf, pfkA, glmM) that control the major competing reactions of GlcNAc synthesis (pentose phosphate pathway (HMP), glycolysis, and peptidoglycan synthesis pathway (PSP)), with the potential to relieve glucose repression and allow the co-utilization of both glucose and xylose. Simultaneous repression of these three genes by CRISPRi improved GlcNAc titer by 13.2% to 17.4 ± 0.47 g/L, with the GlcNAc yield on glucose and xylose showing an 84.1% improvement, reaching 0.42 ± 0.036 g/g. In order to further engineer the synergetic utilization of glucose and xylose, a combinatorial approach was developed based on 27 arrays containing sgRNAs with different repression capacities targeting the three genes. We further optimized the temporal control of the system and found that when 15 g/L xylose was added 6 h after inoculation, the most efficient strain, BNX122, synthesized 20.5 ± 0.85 g/L GlcNAc with a yield of 0.46 ± 0.010 g/g glucose and xylose in shake flask culture. Finally, the GlcNAc titer and productivity in a 3-L fed-batch bioreactor reached 103.1 ± 2.11 g/L and 1.17 ± 0.024 g/L/h, which were 5.0-fold and 2.7-fold of that in shake flask culture, respectively. Taken together, these findings suggest that a CRISPRi-enabled regulation method provides a simple, efficient, and universal way to promote the synergetic utilization of multiple carbon sources by microbial cell factories.

Natural life is encoded by evolvable, DNA-based memory. Recent advances in dynamic genome-engineering technologies, which we collectively refer to as in vivo DNA writing, have opened new avenues for investigating and engineering biology. This Review surveys these technological advances, outlines their prospects and emerging applications, and discusses the features and current limitations of these technologies for building various genetic circuits for processing and recording information in living cells.