scholarly journals In vivo Hepatocyte Genome Manipulation via Intravenous Injection of Genome Editing Components

2020 ◽  
Vol 4 (4) ◽  
Author(s):  
Shingo Nakamura ◽  
◽  
Naoko Ando ◽  
Masayuki Ishihara ◽  
Masahiro Sato
Keyword(s):  
Genome Editing ◽  
Intravenous Injection ◽  
Liver Dysfunction ◽  
Liver Diseases ◽  
Target Genes ◽  
Liver Tumors ◽  
Genetic Manipulation ◽  
Tail Vein ◽  
Short Period ◽  
Wide Range

The liver is a major organ with a wide range of functions, including detoxification, protein synthesis, and bile production. Liver dysfunction causes liver diseases such as hepatic cirrhosis and hepatitis. To explore the pathogenesis of these liver diseases, and the therapeutic agents against them, mice have been widely used as animal models. Genetic manipulation is easy in mice via the administration of nucleic acids (NAs) in the tail-vein. In particular, hydrodynamics-based gene delivery (HGD) is a method based on the introduction of a large volume of NA-containing solution over a short period in the tail-vein. It is recognized as a powerful tool to efficiently transfect hepatocytes. Genome editing, as illustrated by the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9) system, has also been recognized as a powerful tool to manipulate target genes in host genomes. Recently, studies have described the tail-vein-mediated introduction of genome editing components for the generation of liver tumors, correction of mutated genes causing liver dysfunction, and generation of mice with liver disease. More importantly, this HGD method can bypass the need to create mouse progeny carrying the targeted mutation in their germline. In this review, the past and present achievements of liver-targeted manipulation achieved via intravenous injection of genome editing components will be summarized.

scholarly journals Base editing in Streptomyces with Cas9-deaminase fusions

2019 ◽  
Author(s):  
Zhiyu Zhong ◽  
Junhong Guo ◽  
Liang Deng ◽  
Li Chen ◽  
Jian Wang ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Strand Break ◽  
High Fidelity ◽  
Nucleotide Substitutions ◽  
Base Editing ◽  
Dna Double Strand Break ◽  
Repair Template ◽  
Adenine Base

AbstractConventional CRISPR/Cas genetic manipulation has been profitably applied to the genus Streptomyces, the most prolific bacterial producers of antibiotics. However, its reliance on DNA double-strand break (DSB) formation leads to unacceptably low yields of desired recombinants. We have adapted for Streptomyces recently-introduced cytidine base editors (CBEs) and adenine base editors (ABEs) which enable targeted C-to-T or A-to-G nucleotide substitutions, respectively, bypassing DSB and the need for a repair template. We report successful genome editing in Streptomyces at frequencies of around 50% using defective Cas9-guided base editors and up to 100% by using nicked Cas9-guided base editors. Furthermore, we demonstrate the multiplex genome editing potential of the nicked Cas9-guided base editor BE3 by programmed mutation of nine target genes simultaneously. Use of the high-fidelity version of BE3 (HF-BE3) essentially improved editing specificity. Collectively, this work provides a powerful new tool for genome editing in Streptomyces.

scholarly journals Expression of Bacillus ginsengihumi M2.11 bacterial phytase by recombinant Pichia pastoris strains

2021 ◽  
Vol 36 ◽  
pp. 07013
Author(s):  
Daria L. Itkina ◽  
Aliya D. Suleymanova ◽  
Margarita R. Sharipova
Keyword(s):  
Phytic Acid ◽  
Recombinant Proteins ◽  
Genetic Manipulation ◽  
Organic Phosphorus ◽  
Maximum Activity ◽  
Feed Additives ◽  
Animal Nutrition ◽  
Efficient System ◽  
Short Period ◽  
Wide Range

Phytic acid is the main storage form of organic phosphorus. Due to its structural features, phosphorus in phytate is inaccessible for assimilation by animals. Moreover, remaining inaccessible reservoir of phosphorus for animal nutrition, phytic acid is capable of forming insoluble complex salts, which lead to soil and water pollution. Мicrobial enzymes - phytases, capable of decomposing phytic acid to organic phosphorus are being used as feed additives in animal nutrition to solve this problem. Thus, search and development of technologies for the production of enzymes on an industrial scale are the most urgent. Methylotrophic yeast P. pastoris are widely used in biotechnology, as an efficient system for the recombinant proteins expression. They have many advantages, including rapid growth on inexpensive media, a wide range of molecular tools for genetic manipulation in optimizing production processes, they are safe for humans and animals, carry-out many post-translational modifications and produce recombinant proteins intracellularly or extracellularly within a short period of time. It was found that the recombinant P. pastoris strains pPINK-LC-α-MF -phyC, pPINK-HC-α-amyl -phyC, pPINK-LC-α-amyl -phyC, pPINK-HC-α-MF -phyC are able to produce and to secrete B. ginsengihumi bacterial phytase M 2.11 phyC. The maximum activity was observed in the pPINK-LC-α-MF strain – 2.6 (U / mg). Recombinant B. ginsengihumi M 2.11 phytases exhibited high activity in a wide pH range from 2.5 to 9.0. The MF-phyC-HC construction is pH stable. The temperature optimum of all recombinant phytases corresponds to 37 ° C; recombinant phytases retain their activity in the range from -80 to 90C.

scholarly journals Role of the Nrf2-ARE Pathway in Liver Diseases

2013 ◽  
Vol 2013 ◽  
pp. 1-9 ◽  
Author(s):  
Sang Mi Shin ◽  
Ji Hye Yang ◽  
Sung Hwan Ki
Keyword(s):  
Liver Diseases ◽  
Target Genes ◽  
Antioxidant Response ◽  
Related Factor ◽  
Genes Expression ◽  
Wide Range ◽  
Range Of Functions ◽  
Central Organ ◽  
Essential Transcription Factor

The liver is a central organ that performs a wide range of functions such as detoxification and metabolic homeostasis. Since it is a metabolically active organ, liver is particularly susceptible to oxidative stress. It is well documented that liver diseases including hepatitis, fibrosis, cirrhosis, and hepatocellular carcinoma are highly associated with antioxidant capacity. NF-E2-related factor-2 (Nrf2) is an essential transcription factor that regulates an array of detoxifying and antioxidant defense genes expression in the liver. It is activated in response to electrophiles and induces its target genes by binding to the antioxidant response element (ARE). Therefore, the roles of the Nrf2-ARE pathway in liver diseases have been extensively investigated. Studies from several animal models suggest that the Nrf2-ARE pathway collectively exhibits diverse biological functions against viral hepatitis, alcoholic and nonalcoholic liver disease, fibrosis, and cancer via target gene expression. In this review, we will discuss the role of the Nrf2-ARE pathway in liver pathophysiology and the potential application of Nrf2 as a therapeutic target to prevent and treat liver diseases.

scholarly journals Enhanced mating-type switching and sexual hybridization in heterothallic yeast Yarrowia lipolytica

2020 ◽  
Vol 20 (2) ◽  
Author(s):  
Changpyo Han ◽  
Heeun Kwon ◽  
Gyuyeon Park ◽  
Minjeong Jang ◽  
Hye-Jeong Lee ◽  
...  
Keyword(s):  
Yarrowia Lipolytica ◽  
Mating Type ◽  
Genome Engineering ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Industrial Applications ◽  
Single Step ◽  
Knockout Mutant ◽  
Wide Range ◽  
Mating Type Switching

ABSTRACT Yarrowia lipolytica is a non-conventional, heterothallic, oleaginous yeast with wide range of industrial applications. Increasing ploidy can improve advantageous traits for industrial applications including genetic stability, stress resistance, and productivity, but the construction of knockout mutant strains from polyploid cells requires significant effort due to the increased copy numbers of target genes. The goal of this study was to evaluate the effectiveness of a mating-type switching strategy by single-step transformation without a genetic manipulation vestige, and to optimize the conventional method for increasing ploidy (mating) in Y. lipolytica. In this study, mating-type genes in haploid Y. lipolytica cells were scarlessly converted into the opposite type genes by site-specific homologous recombination, and the resulting MATB-type cells were mated at low temperature (22°C) with addition of sodium citrate with each MATA-type haploid cell to yield a MATA/MATB-type diploid strain with genetic information from both parental strains. The results of this study can be used to increase ploidy and for whole genome engineering of a yeast strain with unparalleled versatility for industrial application.

scholarly journals Approaches in the Photosynthetic Production of Sustainable Fuels by Cyanobacteria using Tools of Synthetic Biology

2021 ◽  
Author(s):  
Indrajeet . ◽  
Akhil Rautela ◽  
Sanjay Kumar
Keyword(s):  
Synthetic Biology ◽  
Fossil Fuels ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Biofuel Production ◽  
Genetic Composition ◽  
Cell Factories ◽  
Wide Range ◽  
Photosynthetic Production ◽  
Single Operon

Cyanobacteria, photosynthetic prokaryotic microorganisms having a simple genetic composition are the prospective photoautotrophic cell factories for the production of a wide range of biofuel molecules. Simple genetic composition of cyanobacteria allows effortless genetic manipulation which leads to increased research endeavour from the synthetic biology approach. An improved development of synthetic biology tools, genetic modification methods and advancement in transformation techniques to construct a strain which can contain multiple target genes in single operon will vastly expand the functions that can be used for engineering photosynthetic cyanobacteria for the generation of biofuels. In this review, recent advancements and approaches in synthetic biology tools and biofuel production by metabolically engineered cyanobacteria have been discussed. Various fuel molecules like isoprene, limonene, α-farnesene, squalene, alkanes, butanol and fatty acids which can be a substitute of petroleum and fossil fuels in future have been elaborated.

scholarly journals Recent Advances in Genome Editing Tools in Medical Mycology Research

2021 ◽  
Vol 7 (4) ◽  
pp. 257
Author(s):  
Sanaz Nargesi ◽  
Saeed Kaboli ◽  
Jose Thekkiniath ◽  
Somayeh Heidari ◽  
Fatemeh Keramati ◽  
...  
Keyword(s):  
Genome Editing ◽  
Target Genes ◽  
Genetic Manipulation ◽  
Fungal Infections ◽  
Restriction Enzymes ◽  
Pathogenic Fungi ◽  
Zinc Finger Nucleases ◽  
Medical Mycology ◽  
Double Stranded Dna ◽  
Recent Advances

Manipulating fungal genomes is an important tool to understand the function of target genes, pathobiology of fungal infections, virulence potential, and pathogenicity of medically important fungi, and to develop novel diagnostics and therapeutic targets. Here, we provide an overview of recent advances in genetic manipulation techniques used in the field of medical mycology. Fungi use several strategies to cope with stress and adapt themselves against environmental effectors. For instance, mutations in the 14 alpha-demethylase gene may result in azole resistance in Aspergillusfumigatus strains and shield them against fungicide’s effects. Over the past few decades, several genome editing methods have been introduced for genetic manipulations in pathogenic fungi. Application of restriction enzymes to target and cut a double-stranded DNA in a pre-defined sequence was the first technique used for cloning in Aspergillus and Candida. Genome editing technologies, including zinc-finger nucleases (ZFNs) and transcriptional activator-like effector nucleases (TALENs), have been also used to engineer a double-stranded DNA molecule. As a result, TALENs were considered more practical to identify single nucleotide polymorphisms. Recently, Class 2 type II Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)/Cas9 technology has emerged as a more useful tool for genome manipulation in fungal research.

Cryptotanshinone Suppresses Liver Fibrosis by Attenuating Epithelial-Mesenchymal Transition through Targeting Hedgehog Pathway

2020 ◽  
Vol 20 ◽  
Author(s):  
Shurong Ren ◽  
Qizhen Yue ◽  
Qiubo Wang ◽  
Yanli Zhang ◽  
Bei Zhang
Keyword(s):  
Animal Model ◽  
Liver Fibrosis ◽  
Liver Diseases ◽  
Target Genes ◽  
Epithelial Mesenchymal Transition ◽  
Chronic Liver ◽  
Luciferase Assay ◽  
Induced Expression ◽  
Mesenchymal Transition ◽  
Realtime Pcr

Background: Chronic liver damages from viral infection or alcohol abuse result in liver fibrosis, which is a key pathological event in many types of liver diseases. Discovering new anti-fibrosis agents may provide alternative solutions to manage chronic liver diseases. Methods: We first used CCl4 induced liver fibrosis animal model to evaluate the beneficial effects of Cryptotanshinone (CRY). We next explored target miRNAs regulated by CRY in hepatocytes using microarray. The target miRNA candidate was confirmed with realtime-PCR. We also elucidated the downstream target and pathway directly regulated by the miRNA using luciferase assay, western blotting and Epithelial–Mesenchymal Transition (EMT) markers quantification. Lastly, we confirmed CRY induced expression changes of the target genes in vivo. Results: CRY oral administration markedly alleviated the liver injury caused by CCl4. miRNAs expression profiling and realtime-PCR validation revealed miR-539-3p was directly induced by CRY around 4 folds. The induction of miR-539-3p suppressed SMO expression and antagonized Hedgehog (Hh) pathway. Independently CRY treatment suppressed SMO and inhibited EMT process in hepatocytes. The CRY induced expression changes of both miR-539-3p (~ 2 folds increase) and SMO (~ 60% decrease) in livers were validated in animal model. Conclusion: Our study supported CRY could inhibit liver fibrosis by targeting Hh pathway during EMT. CRY could be used as anti-fibrosis agent candidate for managing chronic liver damages.

scholarly journals Transfer of Plasmid DNA to Clinical Coagulase-Negative Staphylococcal Pathogens by Using a Unique Bacteriophage

2015 ◽  
Vol 81 (7) ◽  
pp. 2481-2488 ◽  
Author(s):  
Volker Winstel ◽  
Petra Kühner ◽  
Bernhard Krismer ◽  
Andreas Peschel ◽  
Holger Rohde
Keyword(s):  
Plasmid Dna ◽  
Genetic Manipulation ◽  
Current Knowledge ◽  
Virulence Determinants ◽  
Coagulase Negative Staphylococci ◽  
Reliable Technique ◽  
Content Type ◽  
Research Activities ◽  
Wide Range ◽  
Genetic Barriers

ABSTRACTGenetic manipulation of emerging bacterial pathogens, such as coagulase-negative staphylococci (CoNS), is a major hurdle in clinical and basic microbiological research. Strong genetic barriers, such as restriction modification systems or clustered regularly interspaced short palindromic repeats (CRISPR), usually interfere with available techniques for DNA transformation and therefore complicate manipulation of CoNS or render it impossible. Thus, current knowledge of pathogenicity and virulence determinants of CoNS is very limited. Here, a rapid, efficient, and highly reliable technique is presented to transfer plasmid DNA essential for genetic engineering to important CoNS pathogens from a uniqueStaphylococcus aureusstrain via a specificS. aureusbacteriophage, Φ187. Even strains refractory to electroporation can be transduced by this technique once donor and recipient strains share similar Φ187 receptor properties. As a proof of principle, this technique was used to delete the alternative transcription factor sigma B (SigB) via allelic replacement in nasal and clinicalStaphylococcus epidermidisisolates at high efficiencies. The described approach will allow the genetic manipulation of a wide range of CoNS pathogens and might inspire research activities to manipulate other important pathogens in a similar fashion.

scholarly journals Biosynthesis of Nature-Inspired Unnatural Cannabinoids

Molecules ◽  
2021 ◽  
Vol 26 (10) ◽  
pp. 2914
Author(s):  
Kevin J. H. Lim ◽  
Yan Ping Lim ◽  
Yossa D. Hartono ◽  
Maybelle K. Go ◽  
Hao Fan ◽  
...  
Keyword(s):  
Synthetic Biology ◽  
Cannabis Sativa ◽  
Genetic Manipulation ◽  
Therapeutic Agents ◽  
Orphan Receptors ◽  
Complex Chemical ◽  
Biological Targets ◽  
Chemical Structures ◽  
Commercial Use ◽  
Wide Range

Natural products make up a large proportion of medicine available today. Cannabinoids from the plant Cannabis sativa is one unique class of meroterpenoids that have shown a wide range of bioactivities and recently seen significant developments in their status as therapeutic agents for various indications. Their complex chemical structures make it difficult to chemically synthesize them in efficient yields. Synthetic biology has presented a solution to this through metabolic engineering in heterologous hosts. Through genetic manipulation, rare phytocannabinoids that are produced in low yields in the plant can now be synthesized in larger quantities for therapeutic and commercial use. Additionally, an exciting avenue of exploring new chemical spaces is made available as novel derivatized compounds can be produced and investigated for their bioactivities. In this review, we summarized the biosynthetic pathways of phytocannabinoids and synthetic biology efforts in producing them in heterologous hosts. Detailed mechanistic insights are discussed in each part of the pathway in order to explore strategies for creating novel cannabinoids. Lastly, we discussed studies conducted on biological targets such as CB1, CB2 and orphan receptors along with their affinities to these cannabinoid ligands with a view to inform upstream diversification efforts.

scholarly journals Citizen views on genome editing: effects of species and purpose

2021 ◽  
Author(s):  
Gesa Busch ◽  
Erin Ryan ◽  
Marina A. G. von Keyserlingk ◽  
Daniel M. Weary
Keyword(s):  
Disease Resistance ◽  
Product Quality ◽  
Genome Editing ◽  
Total Sample ◽  
Moral Foundations ◽  
Perceived Risks ◽  
Public Response ◽  
The Public ◽  
Wide Range ◽  
The Us

AbstractPublic opinion can affect the adoption of genome editing technologies. In food production, genome editing can be applied to a wide range of applications, in different species and with different purposes. This study analyzed how the public responds to five different applications of genome editing, varying the species involved and the proposed purpose of the modification. Three of the applications described the introduction of disease resistance within different species (human, plant, animal), and two targeted product quality and quantity in cattle. Online surveys in Canada, the US, Austria, Germany and Italy were carried out with a total sample size of 3698 participants. Using a between-subject design, participants were confronted with one of the five applications and asked to decide whether they considered it right or wrong. Perceived risks, benefits, and the perception of the technology as tampering with nature were surveyed and were complemented with socio-demographics and a measure of the participants’ moral foundations. In all countries, participants evaluated the application of disease resistance in humans as most right to do, followed by disease resistance in plants, and then in animals, and considered changes in product quality and quantity in cattle as least right to do. However, US and Italian participants were generally more positive toward all scenarios, and German and Austrian participants more negative. Cluster analyses identified four groups of participants: ‘strong supporters’ who saw only benefits and little risks, ‘slight supporters’ who perceived risks and valued benefits, ‘neutrals’ who showed no pronounced opinion, and ‘opponents’ who perceived higher risks and lower benefits. This research contributes to understanding public response to applications of genome editing, revealing differences that can help guide decisions related to adoption of these technologies.

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