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.

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 Homologous recombination-based genome editing by clade F AAVs is inefficient in the absence of a targeted DNA break

2019 ◽  
Author(s):  
Geoffrey L. Rogers ◽  
Hsu-Yu Chen ◽  
Heidy Morales ◽  
Paula M. Cannon
Keyword(s):  
Homologous Recombination ◽  
Progenitor Cells ◽  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Dna Breaks ◽  
Adeno Associated Virus ◽  
Hematopoietic Stem ◽  
Double Stranded Dna ◽  
Dna Break

AbstractAdeno-associated virus (AAV) vectors are frequently used as donor templates for genome editing by homologous recombination. Although modification rates are typically under 1%, they are greatly enhanced by targeted double-stranded DNA breaks (DSBs). A recent report described clade F AAVs mediating high-efficiency homologous recombination-based editing in the absence of DSBs. The clade F vectors included AAV9 and a series isolated from human hematopoietic stem/progenitor cells (HSPCs). We evaluated these vectors by packaging homology donors into AAV9 and an AAVHSC capsid and examining their ability to insert GFP at the CCR5 or AAVS1 loci in human HSPCs and cell lines. As a control we used AAV6, which effectively edits HSPCs, but only when combined with a targeted DSB. Each AAV vector promoted GFP insertion in the presence of matched CCR5 or AAVS1 zinc finger nucleases (ZFNs), but none supported detectable editing in the absence of the nucleases. Rates of editing with ZFNs correlated with transduction efficiencies for each vector, implying no differences in the ability of donor sequences delivered by the different vectors to direct genome editing. Our results therefore do not support that clade F AAVs can perform high efficiency genome editing in the absence of a DSB.

Genome editing of maize

2021 ◽  
pp. 341-376
Author(s):  
Jacob D. Zobrist ◽  
◽  
Morgan McCaw ◽  
Minjeong Kang ◽  
Alan L. Eggenberger ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Target Genes ◽  
Zinc Finger Nucleases ◽  
Maize Genome ◽  
Delivery Methods ◽  
Agricultural Improvement ◽  
Modern Maize ◽  
Associated Proteins ◽  
Short Palindromic Repeat

Developed over thousands of years largely through human intervention, the modern maize genome can now be precisely modified for agricultural improvement and scientific research. This chapter focuses on progress made in recent decades utilizing site-specific nuclease (SSN) technologies in maize genome engineering. Many SSNs, such as meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated proteins (Cas) have been used in maize for both functional analysis and trait improvement. The chapter summarizes the recent innovations related to maize genome editing using SSN technologies, the type of approaches, target genes and traits, and reagent delivery methods. It also discusses the current challenges as well as potential improvements for maize genome engineering protocols.

scholarly journals The Gene Sculpt Suite: a set of tools for genome editing

2019 ◽  
Vol 47 (W1) ◽  
pp. W175-W182 ◽  
Author(s):  
Carla M Mann ◽  
Gabriel Martínez-Gálvez ◽  
Jordan M Welker ◽  
Wesley A Wierson ◽  
Hirotaka Ata ◽  
...  
Keyword(s):  
Genome Editing ◽  
Zinc Finger Nucleases ◽  
Machine Learning Method ◽  
Repair Pathway ◽  
End Joining ◽  
Web Based ◽  
Double Stranded Dna ◽  
Break Site ◽  
Dna Editing ◽  
Genomic Locations

Abstract The discovery and development of DNA-editing nucleases (Zinc Finger Nucleases, TALENs, CRISPR/Cas systems) has given scientists the ability to precisely engineer or edit genomes as never before. Several different platforms, protocols and vectors for precision genome editing are now available, leading to the development of supporting web-based software. Here we present the Gene Sculpt Suite (GSS), which comprises three tools: (i) GTagHD, which automatically designs and generates oligonucleotides for use with the GeneWeld knock-in protocol; (ii) MEDJED, a machine learning method, which predicts the extent to which a double-stranded DNA break site will utilize the microhomology-mediated repair pathway; and (iii) MENTHU, a tool for identifying genomic locations likely to give rise to a single predominant microhomology-mediated end joining allele (PreMA) repair outcome. All tools in the GSS are freely available for download under the GPL v3.0 license and can be run locally on Windows, Mac and Linux systems capable of running R and/or Docker. The GSS is also freely available online at www.genesculpt.org.

scholarly journals Genome Editing: A New Horizon for Oral and Craniofacial Research

2018 ◽  
Vol 98 (1) ◽  
pp. 36-45 ◽  
Author(s):  
N. Yu ◽  
J. Yang ◽  
Y. Mishina ◽  
W.V. Giannobile
Keyword(s):  
Genome Editing ◽  
Genetic Manipulation ◽  
Zinc Finger Nucleases ◽  
Biological Research ◽  
Transcription Activator ◽  
Developmental Defects ◽  
Genetic Manipulations ◽  
Research Fields ◽  
Repair Mechanisms ◽  
Conducting Research

Precise and efficient genetic manipulations have enabled researchers to understand gene functions in disease and development, providing a platform to search for molecular cures. Over the past decade, the unprecedented advancement of genome editing techniques has revolutionized the biological research fields. Early genome editing strategies involved many naturally occurring nucleases, including meganucleases, zinc finger nucleases, and transcription activator-like effector-based nucleases. More recently, the clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated nucleases (CRISPR/Cas) system has greatly enriched genetic manipulation methods in conducting research. Those nucleases generate double-strand breaks in the target gene sequences and then utilize DNA repair mechanisms to permit precise yet versatile genetic manipulations. The oral and craniofacial field harbors a plethora of diseases and developmental defects that require genetic models that can exploit these genome editing techniques. This review provides an overview of the genome editing techniques, particularly the CRISPR/Cas9 technique, for the oral and craniofacial research community. We also discuss the details about the emerging applications of genome editing in oral and craniofacial biology.

scholarly journals Genome Editing by CRISPR-Cas: A Game Change in the Genetic Manipulation of Chlamydomonas

Life ◽  
2020 ◽  
Vol 10 (11) ◽  
pp. 295
Author(s):  
Manel Ghribi ◽  
Serge Basile Nouemssi ◽  
Fatma Meddeb-Mouelhi ◽  
Isabel Desgagné-Penix
Keyword(s):  
Genome Editing ◽  
Genetic Manipulation ◽  
Nuclear Genome ◽  
Industrial Applications ◽  
Zinc Finger Nucleases ◽  
Knock Out ◽  
Sustainable Resources ◽  
Bioinformatic Tools ◽  
Transformation Methods ◽  
Cas A

Microalgae are promising photosynthetic unicellular eukaryotes among the most abundant on the planet and are considered as alternative sustainable resources for various industrial applications. Chlamydomonas is an emerging model for microalgae to be manipulated by multiple biotechnological tools in order to produce high-value bioproducts such as biofuels, bioactive peptides, pigments, nutraceuticals, and medicines. Specifically, Chlamydomonas reinhardtii has become a subject of different genetic-editing techniques adapted to modulate the production of microalgal metabolites. The main nuclear genome-editing tools available today include zinc finger nucleases (ZFNs), transcriptional activator-like effector nucleases (TALENs), and more recently discovered the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas) nuclease system. The latter, shown to have an interesting editing capacity, has become an essential tool for genome editing. In this review, we highlight the available literature on the methods and the applications of CRISPR-Cas for C. reinhardtii genetic engineering, including recent transformation methods, most used bioinformatic tools, best strategies for the expression of Cas protein and sgRNA, the CRISPR-Cas mediated gene knock-in/knock-out strategies, and finally the literature related to CRISPR expression and modification approaches.

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 Central Role of the Trehalose Biosynthesis Pathway in the Pathogenesis of Human Fungal Infections: Opportunities and Challenges for Therapeutic Development

2017 ◽  
Vol 81 (2) ◽  
Author(s):  
Arsa Thammahong ◽  
Srisombat Puttikamonkul ◽  
John R. Perfect ◽  
Richard G. Brennan ◽  
Robert A. Cramer
Keyword(s):  
Stress Responses ◽  
Biosynthetic Pathway ◽  
Fungal Infections ◽  
Pathogenic Fungi ◽  
Antifungal Drugs ◽  
Antifungal Drug ◽  
Medical Mycology ◽  
Content Type ◽  
Trehalose Biosynthesis

SUMMARY Invasive fungal infections cause significant morbidity and mortality in part due to a limited antifungal drug arsenal. One therapeutic challenge faced by clinicians is the significant host toxicity associated with antifungal drugs. Another challenge is the fungistatic mechanism of action of some drugs. Consequently, the identification of fungus-specific drug targets essential for fitness in vivo remains a significant goal of medical mycology research. The trehalose biosynthetic pathway is found in a wide variety of organisms, including human-pathogenic fungi, but not in humans. Genes encoding proteins involved in trehalose biosynthesis are mechanistically linked to the metabolism, cell wall homeostasis, stress responses, and virulence of Candida albicans, Cryptococcus neoformans, and Aspergillus fumigatus. While there are a number of pathways for trehalose production across the tree of life, the TPS/TPP (trehalose-6-phosphate synthase/trehalose-6-phosphate phosphatase) pathway is the canonical pathway found in human-pathogenic fungi. Importantly, data suggest that proteins involved in trehalose biosynthesis play other critical roles in fungal metabolism and in vivo fitness that remain to be fully elucidated. By further defining the biology and functions of trehalose and its biosynthetic pathway components in pathogenic fungi, an opportunity exists to leverage this pathway as a potent antifungal drug target. The goal of this review is to cover the known roles of this important molecule and its associated biosynthesis-encoding genes in the human-pathogenic fungi studied to date and to employ these data to critically assess the opportunities and challenges facing development of this pathway as a therapeutic target.

Sign in / Sign up

Export Citation Format

Share Document