A Hyperthermophilic Argonaute From Ferroglobus placidus With Specificity on Guide Binding Pattern

Argonaute proteins (Agos) from thermophilic archaea are involved in several important processes, such as host defense and DNA replication. The catalytic mechanism of Ago from different microbes with great diversity and genome editing potential is attracting increasing attention. Here, we describe an Argonaute from hyperthermophilic Ferroglobus placidus (FpAgo), with a typical DNA-guided DNA endonuclease activity but adopted with only a short guide 15–20 nt length rather than a broad guide selectivity for reported Agos. FpAgo performed the precise cleavage of phosphodiester bonds between 10 and 11 nt on the target strand (counting from the guide strand) guided strictly by 5′-phosphorylated DNA at temperatures ranging from 75 to 99°C. The cleavage activity was regulated by the divalent cations Mn2+, Mg2+, Co2+, and Ni2+. In addition, FpAgo possesses guide/target mismatch tolerance in the seed region but is sensitive to mismatches in the 3′-guide region. Notably, the EMSA assay revealed that the FpAgo-guide-target ternary complex exhibited a stronger binding affinity for short 15 and 16 nt guide DNAs than longer guides. Moreover, we performed structural modeling analyses that implied the unique PAZ domain of FpAgo for 3′-guide recognition and binding to affect guide length specificity. This study broadens our understanding of thermophilic Agos and paves the way for their use in DNA manipulation.

DNA Manipulation and Single-Molecule Imaging

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

A chimera including a GROWTH-REGULATING FACTOR (GRF) and its cofactor GRF-INTERACTING FACTOR (GIF) increases transgenic plant regeneration efficiency

Genome editing allows precise DNA manipulation, but its potential is limited in many crops by low regeneration efficiencies and few transformable genotypes. Here, we show that expression of a chimeric protein including wheat GROWTH-REGULATING FACTOR 4 (GRF4) and its cofactor GRF-INTERACTING FACTOR 1 (GIF1) dramatically increases the efficiency and speed of regeneration in wheat, triticale and rice and expands the number of transformable wheat genotypes. Moreover, GRF4-GIF1 induces efficient wheat regeneration in the absence of exogenous cytokinins, which facilitates selection of transgenic plants without selectable markers. By combining GRF4-GIF1 and CRISPR-Cas9 technologies, we were able to generate large numbers of edited wheat plants. The GRF4-GIF1 transgenic plants were fertile and without obvious developmental defects, likely due to post-transcriptional regulatory mechanisms operating on GRF4 in adult tissues. Finally, we show that a dicot GRF-GIF chimera improves regeneration efficiency in citrus suggesting that this strategy can be expanded to dicot crops.

An overview of synthetic biology

Synthetic Biology is the combination of basic sciences with engineering. The aim of Synthetic Biology is to create, design, and redesign biological systems and devices to understand biological processes and to achieve useful and sophisticated functionalities to improve human welfare. When the engineering community took part in the discussion for the definition of Synthetic Biology, the idea of extraction and reassembly of “biological parts” along with the principles of abstraction, modularity, and standardization was introduced. Genetic Engineering is one of the many essential tools for synthetic biology, and even though they share the DNA manipulation basis and approach to intervene in the complexity of molecular biology, they differ in many aspects, and the two terms should not be used interchangeably. Some of the applications that have already been done by Synthetic Biology include the production of 1,4-butanediol (BDO), the antimalarial drug artemisinin, and the anticancer compound taxol. The potential of Synthetic Biology to design new genomes without immediate biological ancestry has raised ontological, political, economic, and ethical concerns based on the possibility that synthetic biology may be intrinsically unethical.

Review. Development, Applications, Benefits, Challenges and Limitations of the New Genome Engineering Technique. An Update Study

We assume that the CRISPR Cas9 theory must be delimited by applicability, because the consequences of long term DNA manipulation remain unknown. Moreover, the irreversibility of this procedure should instigate researchers to reserved opinions.Usefulness as well as benefits of CRISPR Cas9 made it one of the most popular and used genome editing technique. But with its huge potential, ethical and safety concerns emerge. Therefore, before continuing research in this direction we should have a well organized system that is able to make that differentiation between research and reproduction. However we truly believe in the future of genetic engineering and with the CRISPR-Cas9 system we expect that the opportunity of treating now so called incurable diseases arises. Time is all we need.

Open source approaches to establishingRoseobacterclade bacteria as synthetic biology chassis for biogeoengineering

Aim.The nascent field of bio-geoengineering stands to benefit from synthetic biologists’ efforts to standardise, and in so doing democratise, biomolecular research methods.Roseobacterclade bacteria comprise 15–20% of oceanic bacterio-plankton communities, making them a prime candidate for establishment of synthetic biology chassis for bio-geoengineering activities such as bioremediation of oceanic waste plastic. Developments such as the increasing affordability of DNA synthesis and laboratory automation continue to foster the establishment of a global ‘do-it-yourself’ research community alongside the more traditional arenas of academe and industry. As a collaborative group of citizen, student and professional scientists we sought to test the following hypotheses: (i) that an incubator capable of cultivating bacterial cells can be constructed entirely from non-laboratory items, (ii) that marine bacteria from theRoseobacterclade can be established as a genetically tractable synthetic biology chassis using plasmids conforming to the BioBrickTMstandard and finally, (iii) that identifying and subcloning genes from aRoseobacterclade species can readily by achieved by citizen scientists using open source cloning and bioinformatic tools.Method.We cultivated threeRoseobacterspecies,Roseobacter denitrificans,Oceanobulbus indolifexandDinoroseobacter shibae. For each species we measured chloramphenicol sensitivity, viability over 11 weeks of glycerol-based cryopreservation and tested the effectiveness of a series of electroporation and heat shock protocols for transformation using a variety of plasmid types. We also attempted construction of an incubator-shaker device using only publicly available components. Finally, a subgroup comprising citizen scientists designed and attempted a procedure for isolating the cold resistanceanf1gene fromOceanobulbus indolifexcells and subcloning it into a BioBrickTMformatted plasmid.Results.All species were stable over 11 weeks of glycerol cryopreservation, sensitive to 17 µg/mL chloramphenicol and resistant to transformation using the conditions and plasmids tested. An incubator-shaker device, ‘UCLHack-12’ was assembled and used to cultivate sufficient quantity ofOceanobulbus indolifexcells to enable isolation of theanf1gene and its subcloning into a plasmid to generate the BioBrickTMBBa_K729016.Conclusion.The process of ‘de-skilling’ biomolecular techniques, particularly for relatively under-investigated organisms, is still on-going. However, our successful cell growth and DNA manipulation experiments serve to indicate the types of capabilities that are now available to citizen scientists. Science democratised in this way can make a positive contribution to the debate around the use of bio-geoengineering to address oceanic pollution or climate change.