CRISPR-Cas9: Role in Processing of Modular Metabolic Engineered Bio-Based Products

Biogenetic engineering is a significant technology to sensibly manage microbial metabolic product factories. Genome modification methods for efficiently controlling and modifying genes at the genome level have progressed in biogenetic engineering during the last decade. CRISPR is genome editing technology that allows for the modification of organisms’ genomes. CRISPR and its related RNA-guided endonuclease are versatile advanced immune system frameworks for defending against foreign DNA and RNAs. CRISPR is efficient, accessible, and trustworthy genomic modification tool in unparalleled resolution. At present, CRISPR-Cas9 method is expanded to industrially manipulate cells. Metabolically modified organisms are quickly becoming interested in the production of different bio-based components. Here, chapter explore about the control productivity of targeted biomolecules in divergent cells based on the use of different CRISPR-related Cas9.

Citric acid/β-alanine carbon dots as a novel tool for delivery of plasmid DNA into E. coli cells

Successful delivery of plasmid DNA into the microbial cells is fundamental in recombinant DNA technology. Natural bacterial transformation is limited to only certain species due in part to the repulsive forces between negatively charged DNA and bacterial membranes. Most common method of DNA delivery into bacteria is artificial transformation through heat shock and electroporation. These methods require sophisticated instruments and tedious steps in preparation of competent cells. Transformation by conjugation is also not applicable to all plasmids. Nanoparticles have been used successfully in therapeutics for drug delivery into animal cells. They are starting to gain popularity in plant sciences as novel DNA nano carriers. Despite their promise as tool for DNA delivery, their use in microbial cell transformation has not been reported yet. Here we report the synthesis of carbon dots (CDs) from citric acid and β-alanine and their use in DNA delivery into E. coli cells. CDs were fabricated using microwave assisted synthesis. Plasmids carrying RFP reporter and ampicillin resistance genes were transferred to bacterial cells and further confirmed using polymerase chain reaction. Our findings indicate that CDs can be used successfully for delivery of foreign DNA of up to 10 kb into E. coli. We have demonstrated the use of β-alanine/citric acid carbon dots as nanocarriers of DNA into E. coli cells and identified their limitation in terms of the size of plasmid DNA they could carry. Use of these carbon dots is a novel method in foreign DNA delivery into bacterial cells and have a potential for the transformation of resistant organism for which there is still no reliable DNA delivery systems.

An siRNA-guided ARGONAUTE protein directs RNA polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without pre-existing DNA methylation. We find that Pol V follows and is dependent on the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of pre-existing DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

Ribosomal protein S1 plays a critical role in horizontal gene transfer by mediating the expression of foreign mRNAs

The emergence of multi-antibiotic resistant bacteria is one of the largest threats to global heath. This rise is due to the genomic plasticity of bacteria, allowing rapid acquisition of antibiotic resistance through the uptake of foreign DNA (i.e. horizontal gene transfer, HGT). This genomic plasticity is not limited to DNA from bacteria, highly divergent (trans-kingdom) mRNA have been reported to drive translation in E. coli. Trans-kingdom activity has been attributed to mRNA tertiary structure suggesting the bacterial translation machinery bottle-necks HGT, restricting the expression of foreign DNA. However, here we show that tertiary structure is not responsible for ribosome recruitment and that the translation efficiency is dependent on ribosomal protein S1 and an A-rich Shine-Dalgarno-like element. The S1-facilitated ability of ribosomes to identify and exploit A-rich sequences in foreign RNA highlights the important role that S1 plays in horizontal gene transfer, the robustness of canonical prokaryotic translation, and bacterial evolution.

Sensing Stemness

Purpose of Review Hematopoietic stem cells (HSCs) are formed embryonically during a dynamic developmental process and later reside in adult hematopoietic organs in a quiescent state. In response to their changing environment, HSCs have evolved diverse mechanisms to cope with intrinsic and extrinsic challenges. This review intends to discuss how HSCs and other stem cells co-opted DNA and RNA innate immune pathways to fine-tune developmental processes. Recent Findings Innate immune receptors for nucleic acids like the RIG-I-like family receptors and members of DNA sensing pathways are expressed in HSCs and other stem cells. Even though the “classic” role of these receptors is recognition of foreign DNA or RNA from pathogens, it was recently shown that cellular transposable element (TE) RNA or R-loops activate such receptors, serving as endogenous triggers of inflammatory signaling that can shape HSC formation during development and regeneration. Summary Endogenous TEs and R-loops activate RNA and DNA sensors, which trigger distinct inflammatory signals to fine-tune stem cell decisions. This phenomenon could have broad implications for diverse somatic stem cells, for a variety of diseases and during aging.

Nuclear soluble cGAS senses DNA virus infection

The cytosolic DNA sensor cGAS detects foreign DNA from pathogens or self-DNA from cellular damage and instigates type I interferon (IFN) expression. Recent studies find that cGAS also localizes in the nucleus and binds the chromatin. Despite how cGAS is inhibited in the nucleus is well elucidated, whether nuclear cGAS participates in DNA sensing is not clear. Here, we report that herpes simplex virus 1 (HSV-1) infection caused the release of cGAS from the chromatin into the nuclear soluble fraction. Like its cytosolic counterpart, the leaked nuclear soluble cGAS could sense viral DNA, produce cGAMP, and induce mRNA expression of type I IFN and interferon-stimulated genes. Furthermore, the nuclear cGAS limited HSV-1 infection. Taken together, our study demonstrates that HSV-1 infection releases cGAS from the chromatin tethering and, in turn, the nuclear soluble cGAS activates type I IFN production.

Involvement of the Histone-Like Nucleoid Structuring Protein (H-NS) in Acinetobacter baumannii’s Natural Transformation

Most Acinetobacter baumannii strains are naturally competent. Although some information is available about factors that enhance or reduce the frequency of the transformation of this bacterium, the regulatory elements and mechanisms are barely understood. In this article, we describe studies on the role of the histone-like nucleoid structuring protein, H-NS, in the regulation of the expression of genes related to natural competency and the ability to uptake foreign DNA. The expression levels of the natural transformation-related genes pilA, pilT, pilQ, comEA, comEC, comF, and drpA significantly increased in a Δhns derivative of A. baumannii A118. The complementation of the mutant with a recombinant plasmid harboring hns restored the expression levels of six of these genes (pilT remained expressed at high levels) to those of the wild-type strain. The transformation frequency of the A. baumannii A118 Δhns strain was significantly higher than that of the wild-type. Similar, albeit not identical, there were consequences when hns was deleted from the hypervirulent A. baumannii AB5075 strain. In the AB5075 complemented strain, the reduction in gene expression in a few cases was not so pronounced that it reached wild-type levels, and the expression of comEA was enhanced further. In conclusion, the expression of all seven transformation-related genes was enhanced after deleting hns in A. baumannii A118 and AB5075, and these modifications were accompanied by an increase in the cells’ transformability. The results highlight a role of H-NS in A. baumannii’s natural competence.

An siRNA-guided Argonaute protein directs RNA Polymerase V to initiate DNA methylation

In mammals and plants, cytosine DNA methylation is essential for the epigenetic repression of transposable elements and foreign DNA. In plants, DNA methylation is guided by small interfering RNAs (siRNAs) in a self-reinforcing cycle termed RNA-directed DNA methylation (RdDM). RdDM requires the specialized RNA Polymerase V (Pol V), and the key unanswered question is how Pol V is first recruited to new target sites without preexisting DNA methylation. We find that Pol V follows and is dependent upon the recruitment of an AGO4-clade ARGONAUTE protein, and any siRNA can guide the ARGONAUTE protein to the new target locus independent of preexisting DNA methylation. These findings reject long-standing models of RdDM initiation and instead demonstrate that siRNA-guided ARGONAUTE targeting is necessary, sufficient and first to target Pol V recruitment and trigger the cycle of RdDM at a transcribed target locus, thereby establishing epigenetic silencing.

Let me upgrade you: Impact of mobile genetic elements on enterococcal adaptation and evolution

Enterococci are Gram-positive bacteria that have evolved to thrive as both commensals and pathogens, largely due to their accumulation of mobile genetic elements via horizontal gene transfer. Common agents of horizontal gene transfer include plasmids, transposable elements, and temperate bacteriophages. These vehicles have facilitated the evolution of the enterococci, specifically Enterococcus faecalis and Enterococcus faecium , into multidrug resistant hospital-acquired pathogens. On the other hand, commensal strains of Enterococcus harbor CRISPR-Cas systems that prevent the acquisition of foreign DNA, restricting the accumulation of mobile genetic elements. In this review we discuss enterococcal mobile genetic elements by highlighting their contributions to bacterial fitness, examine the impact of CRISPR-Cas on their acquisition, and identify key areas of research that can improve our understanding of enterococcal evolution and ecology.