An inexpensive anaerobic chamber for the genetic manipulation of strictly anaerobic bacteria.

Strictly anaerobic bacteria are important to both human health and industrial usage. These bacteria are sensitive to oxygen, therefore, it is preferable to manipulate these microbes in an anaerobic chamber. However, commercial anaerobic chambers (CACs) are expensive, making them less accessible to scientists with a limited budget, especially to those in developing countries. The high price of commercial chambers has hindered, at least partially, the progress of research on anaerobes in developing countries. In the research presented here, we developed an inexpensive and reliable anaerobic chamber and successfully achieved routine maintenance of eleven strictly anaerobic bacterial strains. Furthermore, genetic manipulation examples have been set for both Clostridioidesdifficile 630 and Clostridiumbeijerinckii NCIMB 8052 strains to validate that the chamber could applied to advanced genetic engineering of strictly anaerobes. C. difficile and C. beijerinckii were both genetically manipulated in this chamber, showing it's utility for the genetic engineering of anaerobes. Most importantly, the anaerobic chamber was 76% - 88% less expensive than a CACs and has similar functionality with regards to the cultivation and manipulation of strictly anaerobic bacteria. The anaerobic chamber described in this study will promote the research of anaerobes in developing counties and scientists who have limited research budgets.

Detection of CRISPR-Cas9-Mediated Mutations Using a Carbon Nanotube-Modified Electrochemical Genosensor.

The CRISPR-Cas9 system has facilitated the genetic modification of various model organisms and cell lines. The outcomes of any CRISPR-Cas9 assay should be investigated to ensure/improve the precision of genome engineering. In this study, carbon nanotube-modified disposable pencil graphite electrodes (CNT/PGEs) were used to develop a label-free electrochemical nanogenosensor for the detection of point mutations generated in the genome by using the CRISPR-Cas9 system. Carbodiimide chemistry was used to immobilize the 5'-aminohexyl-linked inosine-substituted probe on the surface of the sensor. After hybridization between the target sequence and probe at the sensor surface, guanine oxidation signals were monitored using differential pulse voltammetry (DPV). Optimization of the sensitivity of the nanogenoassay resulted in a lower detection limit of 213.7 nM. The nanogenosensor was highly specific for the detection of the precisely edited DNA sequence. This method allows for a rapid and easy investigation of the products of CRISPR-based gene editing and can be further developed to an array system for multiplex detection of different-gene editing outcomes.

Setting Up an Automated Biomanufacturing Laboratory.

Laboratory automation is a key enabling technology for genetic engineering that can lead to higher throughput, more efficient and accurate experiments, better data management and analysis, decrease in the DBT (Design, Build, and Test) cycle turnaround, increase of reproducibility, and savings in lab resources. Choosing the correct framework among so many options available in terms of software, hardware, and skills needed to operate them is crucial for the success of any automation project. This chapter explores the multiple aspects to be considered for the solid development of a biofoundry project including available software and hardware tools, resources, strategies, partnerships, and collaborations in the field needed to speed up the translation of research results to solve important society problems.

Rapid and highly efficient genomic engineering with a novel iEditing device for programming versatile extracellular electron transfer of electroactive bacteria.

The advances in synthetic biology bring exciting new opportunities to reprogram microorganisms with novel functionalities for environmental applications. For real-world applications, a genetic tool that enables genetic engineering in a stably genomic inherited manner is greatly desired. In this work, we design a novel genetic device for rapid and efficient genome engineering based on the intron-encoded homing-endonuclease empowered genome editing (iEditing). The iEditing device enables rapid and efficient genome engineering in Shewanella oneidensis MR-1, the representative strain of the electroactive bacteria group. Moreover, combining with the Red or RecET recombination system, the genome-editing efficiency was greatly improved, up to approximately 100%. Significantly, the iEditing device itself is eliminated simultaneously when genome editing occurs, thereby requiring no follow-up to remove the encoding system. Then, we develop a new extracellular electron transfer (EET) engineering strategy by programming the parallel EET systems to enhance versatile EET. The engineered strains exhibit sufficiently enhanced electron output and pollutant reduction ability. Furthermore, this device has demonstrated its great potential to be extended for genome editing in other important microbes. This work provides a useful and efficient tool for the rapid generation of synthetic microorganisms for various environmental applications.

Reflexiones ético-teológicas ante la edición genética / Genetic edition and theological ethics

El artículo busca anticipar los desafíos que se vislumbran hacia el futuro una vez que se perfeccione la técnica de la edición genética. En primer lugar, se encuadra la edición genética en el marco de la especificidad de la tecnología moderna. En segundo lugar, se plantea cómo el contraste de lo natural con lo artificial ilumina el problema que significa pretender sustituir lo natural, en el sentido de lo dado, con lo proyectado por la tecnología. Posteriormente, después de constatar el carácter incipiente de la reflexión teológica sobre la técnica, se plantea la convergencia de la idea de creación con un respeto por los equilibrios naturales, propios de la sensibilidad ecológica contemporánea. Se muestra así, que la propuesta cristiana no es la tecnofobia, sino la integración de la técnica -como elemento central de la cultura que desarrolla el ser humano- con la naturaleza y la asunción del carácter limitado de las realidades naturales, incluyendo al mismo ser humano. De este modo, la vulnerabilidad, como realización de esta finitud, es la característica que nos iguala y nos exige el reconocimiento de nuestra dignidad, mucho más que la realización de un ideal de perfección tecnológicamente mediado, aunque fuera accesible para todos

This paper tries to look forward to the incoming ethical challenges related with genetic editing. It begins with contextualizing genetic edition within the specific nature of modern technology. Afterwards it presents the contrast between natural beings and artifacts that sheds light for answering the question about the real possibility of replacing natural beings, as they are, with technologically projected living beings. In the third place, after acknowledging the scarce attention given by contemporary theology to technology, it shows the convergence of the Christian concept of creation with the respect for balance in nature, as most part of the contemporaty ecological sensibility upholds. Building on this common ground it shows that the Christian attitude towards technology is not technofobical but the integration of technology -a central element of contemporary culture- with nature, accepting the limitation of any natural being including mankind. In this way, vulnerability, as a visible consequence of this finitude, is the very attribute of human beings that makes ourselves equal and requieres recongnition of our common dignity, way over the idea of acquiring an ideal perfection through technology, even if it was accesible to all

Transformation improvement with the Standardized Pressure Agrobacterium Infiltration Device (SPAID).

OBJECTIVE: The treatment of plant tissue with Agrobacterium tumefaciens is often a critical first step to both stable and transient plant transformation. In both applications bacterial suspensions are oftentimes physically introduced into plant tissues using hand-driven pressure from a needleless syringe. While effective, this approach has several drawbacks that limit reproducibility. Pressure must be provided with the syringe perfectly perpendicular to the tissue surface. The researcher must also attempt to provide even and consistent pressure, both within and between experimental replicates. These factors mean that the procedures do not always translate well between research groups or biological replicates. RESULTS: We have devised a method to introduce Agrobacterium suspensions into plant leaves with greater reproducibility. Using a decommissioned dissecting microscope as an armature, a syringe body with the bacterial suspension is mounted to the nosepiece. Gentle, even pressure is applied by rotating the focus knob. The treatment force is measured using a basic kitchen scale. The development of the Standardized Pressure Agrobacterium Infiltration Device (SPAID) provides a means to deliver consistent amounts of bacterial suspensions into plant tissues with the goal of increasing reproducibility between replicates and laboratories.

Repurposing Macromolecule Delivery Tools for Plant Genetic Modification in the Era of Precision Genome Engineering.

Efficient delivery of macromolecules into plant cells and tissues is important for both basic research and biotechnology product applications. In transgenic research, the goal is to deliver DNA molecules into regenerable cells and stably integrate them into the genome. Over the past 40 years, many macromolecule delivery methods have been studied. To generate transgenic plants, particle bombardment and Agrobacterium-mediated transformation are the methods of choice for DNA delivery. The rapid advance of genome editing technologies has generated new requirements on large biomolecule delivery and at the same time reinvigorated the development of new transformation technologies. Many of the gene delivery options that have been studied before are now being repurposed for delivering genome editing machinery for various applications. This article reviews the major progress in the development of tools for large biomolecule delivery into plant cells in the new era of precision genome engineering.

Protocol for Agrobacterium-Mediated Transformation and Transgenic Plant Production of Switchgrass.

Switchgrass (Panicum virgatum L.) is one of the most important bioenergy crops for lignocellulose ethanol production. Molecular breeding provides a powerful tool to supplement conventional switchgrass breeding by introducing or editing genes of interest. In this chapter, we describe Agrobacterium tumefaciens-mediated transformation protocols for lowland tetraploid switchgrass cultivar Alamo.

DNA Break Repair in Plants and Its Application for Genome Engineering.

Genome engineering is a biotechnological approach to precisely modify the genetic code of a given organism in order to change the context of an existing sequence or to create new genetic resources, e.g., for obtaining improved traits or performance. Efficient targeted genome alterations are mainly based on the induction of DNA double-strand breaks (DSBs) or adjacent single-strand breaks (SSBs). Naturally, all organisms continuously have to deal with DNA-damaging factors challenging the genetic integrity, and therefore a wide range of DNA repair mechanisms have evolved. A profound understanding of the different repair pathways is a prerequisite to control and enhance targeted gene modifications. DSB repair can take place by nonhomologous end joining (NHEJ) or homology-dependent repair (HDR). As the main outcome of NHEJ-mediated repair is accompanied by small insertions and deletions, it is applicable to specifically knock out genes or to rearrange linkage groups or whole chromosomes. The basic requirement for HDR is the presence of a homologous template; thus this process can be exploited for targeted integration of ectopic sequences into the plant genome. The development of different types of artificial site-specific nucleases allows for targeted DSB induction in the plant genome. Such synthetic nucleases have been used for both qualitatively studying DSB repair in vivo with respect to mechanistic differences and quantitatively in order to determine the role of key factors for NHEJ and HR, respectively. The conclusions drawn from these studies allow for a better understanding of genome evolution and help identifying synergistic or antagonistic genetic interactions while supporting biotechnological applications for transiently modifying the plant DNA repair machinery in favor of targeted genome engineering.

Gene Stacking in Plants Through the Application of Site-Specific Recombination and Nuclease Activity.

Biotechnology methods for inserting genes one by one or as a block of fragment into plant genomes are needed to introduce valuable traits into crop varieties. Insertion of multiple genes into a single site, called as molecular stacking, is important to allow co-inheritance of the genes into the progeny. Generally, two approaches are available for creating gene stacks: nuclease-induced targeted gene integration into native sites and recombinase-mediated gene integration into the engineered sites. The recombinase application is attractive as several recombinases show high efficiency and precision in plant genomes. This chapter describes a gene stacking method based on the use of Cre-lox site-specific recombination system to integrate genes into the engineered sites and nucleases to delete selection genes leading to stacking of traits into a single genomic site. High efficiency and precision, and undetectable off-target effects of Cre-lox in a number of plant species, make it an attractive tool for complex applications such as gene stacking.

Plant Biotechnology Applications of Zinc Finger Technology.

With ever-increasing genomic information combined with modern tools for genome modification, we are entering a new era of plant biotechnology. One major tool used for genome modification is the zinc finger nuclease (ZFN). Here, we discuss how ZFNs have proven useful in many genome modification applications. In order to remove the function of a gene or genes, targeted mutagenesis using ZFNs has been readily demonstrated creating numerous gene knockouts, and gene deletion has been demonstrated with removal of gene segments both native and transgenic up to 9 Mb. Applications for gain of function have also been demonstrated. Precision gene editing using ZFNs has resulted in the development of herbicide tolerance, and numerous forms of targeted gene addition have been exhibited. In addition to genome modification, this chapter also describes the use of zinc finger protein transcription factors (ZFP-TFs) for gene regulation in order to provide useful modification of gene expression resulting in altered phenotypes.

[Application of machine learning in the CRISPR/Cas9 system].

The third generation of the CRISPR/Cas9-mediated genome fixed-point editing technology has been widely used in the field of gene editing and gene expression regulation. How to improve the on-target efficiency and specificity of this system, as well as reduce its off-target effects are always the bottleneck in its development. Machine learning provides novel methods to the problems of the CRISPR/Cas9 system, and CRISPR/Cas9-based machine learning has recently become a very hot research topic. In this review, we firstly outline the mechanism of the CRISPR/Cas9 system. Subsequently, we elaborate the current issues of CRISPR/Cas9, including low efficiency and potential off-target effects, and sequence-recognizing limitation from protospacer adjacent motif (PAM). Finally, we summarize the applications of methods within the machine learning framework for optimizing the CRISPR/Cas9 system, such as optimized single-guide RNA (sgRNA) design, CRISPR/Cas9 cleavage efficiency prediction, off-target effects evaluation, gene knock-out as well as high-throughput functional genetic screening and prospects for development.

Silkworm silk-based materials and devices generated using bio-nanotechnology.

Silks are natural fibrous protein polymers that are spun by silkworms and spiders. Among silk variants, there has been increasing interest devoted to the silkworm silk of B. mori, due to its availability in large quantities along with its unique material properties. Silk fibroin can be extracted from the cocoons of the B. mori silkworm and combined synergistically with other biomaterials to form biopolymer composites. With the development of recombinant DNA technology, silks can also be rationally designed and synthesized via genetic control. Silk proteins can be processed in aqueous environments into various material formats including films, sponges, electrospun mats and hydrogels. The versatility and sustainability of silk-based materials provides an impressive toolbox for tailoring materials to meet specific applications via eco-friendly approaches. Historically, silkworm silk has been used by the textile industry for thousands of years due to its excellent physical properties, such as lightweight, high mechanical strength, flexibility, and luster. Recently, due to these properties, along with its biocompatibility, biodegradability and non-immunogenicity, silkworm silk has become a candidate for biomedical utility. Further, the FDA has approved silk medical devices for sutures and as a support structure during reconstructive surgery. With increasing needs for implantable and degradable devices, silkworm silk has attracted interest for electronics, photonics for implantable yet degradable medical devices, along with a broader range of utility in different device applications. This Tutorial review summarizes and highlights recent advances in the use of silk-based materials in bio-nanotechnology, with a focus on the fabrication and functionalization methods for in vitro and in vivo applications in the field of tissue engineering, degradable devices and controlled release systems.

Time to Get Serious about Measurement in Synthetic Biology.

For synthetic biology to mature, composition of devices into functional systems must become routine. This requires widespread adoption of comparable and replicable units of measurement. Interlaboratory studies organized through the International Genetically Engineered Machine (iGEM) competition show that fluorescence can be calibrated with simple, low-cost protocols, so fluorescence should no longer be published without units.

Microinjection of Antisense Morpholinos, CRISPR/Cas9 RNP, and RNA/DNA into Zebrafish Embryos.

In this chapter, we describe a stepwise protocol of microinjection. Using this method, antisense morpholinos, CRISPR-Cas9 ribonucleoprotein complexes, capped mRNA, and DNA can be delivered into fertilized zebrafish eggs to manipulate gene expression during development. This protocol can also be adapted for microinjection in other fish and amphibian species.

Imaging Proteolytic Activities in Mouse Models of Cancer.

Proteases are "protein-cleaving" enzymes, which, in addition to their non-specific degrading function, also catalyze the highly specific and regulated process of proteolytic processing, thus regulating multiple biological functions. Alterations in proteolytic activity occur during pathological conditions such as cancer. One of the major deregulated classes of proteases in cancer is caspases, the proteolytic initiators and mediators of the apoptotic machinery. The ability to image apoptosis noninvasively in living cells and animal models of cancer can not only provide new insight into the biological basis of the disease but can also be used as a quantitative tool to screen and evaluate novel therapeutic strategies. Optical molecular imaging such as bioluminescence-based genetically engineered biosensors has been developed in our laboratory and exploited to study protease activity in animal models with a high signal to noise. Using the circularly permuted form of firefly luciferase, we have developed a reporter for Caspase 3/7, referred to as Caspase 3/7 GloSensor. Here, we discuss the use of the Caspase 3/7 GloSensor for imaging apoptotic activity in mouse xenografts and genetically engineered mouse models of cancer and present the potential of this powerful platform technology to image the proteolytic activity of numerous other proteases.

A Comparison of Techniques to Evaluate the Effectiveness of Genome Editing.

Genome editing using engineered nucleases (meganucleases, zinc finger nucleases, transcription activator-like effector nucleases) has created many recent breakthroughs. Prescreening for efficiency and specificity is a critical step prior to using any newly designed genome editing tool for experimental purposes. The current standard screening methods of evaluation are based on DNA sequencing or use mismatch-sensitive endonucleases. They can be time-consuming and costly or lack reproducibility. Here, we review and critically compare standard techniques with those more recently developed in terms of reliability, time, cost, and ease of use.

Sequence-based prediction of permissive stretches for internal protein tagging and knockdown.

BACKGROUND: Internal tagging of proteins by inserting small functional peptides into surface accessible permissive sites has proven to be an indispensable tool for basic and applied science. Permissive sites are typically identified by transposon mutagenesis on a case-by-case basis, limiting scalability and their exploitation as a system-wide protein engineering tool. METHODS: We developed an apporach for predicting permissive stretches (PSs) in proteins based on the identification of length-variable regions (regions containing indels) in homologous proteins. RESULTS: We verify that a protein's primary structure information alone is sufficient to identify PSs. Identified PSs are predicted to be predominantly surface accessible; hence, the position of inserted peptides is likely suitable for diverse applications. We demonstrate the viability of this approach by inserting a Tobacco etch virus protease recognition site (TEV-tag) into several PSs in a wide range of proteins, from small monomeric enzymes (adenylate kinase) to large multi-subunit molecular machines (ATP synthase) and verify their functionality after insertion. We apply this method to engineer conditional protein knockdowns directly in the Escherichia coli chromosome and generate a cell-free platform with enhanced nucleotide stability. CONCLUSIONS: Functional internally tagged proteins can be rationally designed and directly chromosomally implemented. Critical for the successful design of protein knockdowns was the incorporation of surface accessibility and secondary structure predictions, as well as the design of an improved TEV-tag that enables efficient hydrolysis when inserted into the middle of a protein. This versatile and portable approach can likely be adapted for other applications, and broadly adopted. We provide guidelines for the design of internally tagged proteins in order to empower scientists with little or no protein engineering expertise to internally tag their target proteins.

Genetic Constructor: An Online DNA Design Platform.

Genetic Constructor is a cloud Computer Aided Design (CAD) application developed to support synthetic biologists from design intent through DNA fabrication and experiment iteration. The platform allows users to design, manage, and navigate complex DNA constructs and libraries, using a new visual language that focuses on functional parts abstracted from sequence. Features like combinatorial libraries and automated primer design allow the user to separate design from construction by focusing on functional intent, and design constraints aid iterative refinement of designs. A plugin architecture enables contributions from scientists and coders to leverage existing powerful software and connect to DNA foundries. The software is easily accessible and platform agnostic, free for academics, and available in an open-source community edition. Genetic Constructor seeks to democratize DNA design, manufacture, and access to tools and services from the synthetic biology community.