scholarly journals CRISPR-Based Genetic Manipulation of Candida Species: Historical Perspectives and Current Approaches

2021 ◽  
Vol 2 ◽  
Author(s):  
Deeva Uthayakumar ◽  
Jehoshua Sharma ◽  
Lauren Wensing ◽  
Rebecca S. Shapiro
Keyword(s):  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Single Point ◽  
Antifungal Drug ◽  
Sexual Cycle ◽  
Single Point Mutation ◽  
Functional Genomic ◽  
Historical Perspectives ◽  
Genomic Studies

The Candida genus encompasses a diverse group of ascomycete fungi that have captured the attention of the scientific community, due to both their role in pathogenesis and emerging applications in biotechnology; the development of gene editing tools such as CRISPR, to analyze fungal genetics and perform functional genomic studies in these organisms, is essential to fully understand and exploit this genus, to further advance antifungal drug discovery and industrial value. However, genetic manipulation of Candida species has been met with several distinctive barriers to progress, such as unconventional codon usage in some species, as well as the absence of a complete sexual cycle in its diploid members. Despite these challenges, the last few decades have witnessed an expansion of the Candida genetic toolbox, allowing for diverse genome editing applications that range from introducing a single point mutation to generating large-scale mutant libraries for functional genomic studies. Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 technology is among the most recent of these advancements, bringing unparalleled versatility and precision to genetic manipulation of Candida species. Since its initial applications in Candida albicans, CRISPR-Cas9 platforms are rapidly evolving to permit efficient gene editing in other members of the genus. The technology has proven useful in elucidating the pathogenesis and host-pathogen interactions of medically relevant Candida species, and has led to novel insights on antifungal drug susceptibility and resistance, as well as innovative treatment strategies. CRISPR-Cas9 tools have also been exploited to uncover potential applications of Candida species in industrial contexts. This review is intended to provide a historical overview of genetic approaches used to study the Candida genus and to discuss the state of the art of CRISPR-based genetic manipulation of Candida species, highlighting its contributions to deciphering the biology of this genus, as well as providing perspectives for the future of Candida genetics.

Multilevel Evolution: The Fate of Duplicated Genes

2002 ◽  
Vol 216 (1) ◽  
Author(s):  
Paulien Hogeweg
Keyword(s):  
Information Integration ◽  
Large Scale ◽  
Evolutionary Dynamics ◽  
Single Point ◽  
Gene Content ◽  
Minor Role ◽  
Single Point Mutation ◽  
Duplicated Genes ◽  
A Genome ◽  
Multilevel Evolution

Biological evolution is a multilevel process and should be studied as such. A first, important step in studying evolution in this way has been the work of Peter Schuster and co-workers on RNA evolution. For RNA the genotype-phenotype mapping can be calculated explicitly. The resulting evolutionary dynamics is dominated by neutral paths, and the potential of major change by a single point mutation.Examining whole genomes, of which about 60 are now available, we see that gene content of genomes is changing relatively rapidly: gene duplication, gene loss and gene generation is ubiquitous. In fact, it seems that point-mutations play a relatively minor role, relative to changes in gene regulation and gene content in adaptive evolution.Large scale micro-array studies, in which the expression of every gene can be measured simultaneously, give a first glimpse of the `division of labor´ between duplicated genes. A preliminary analysis suggests that differential expression is often the primary event which allows duplicated genes to be maintained in a genome, but alternate routes also exist, most notably on the one hand the mere need of a lot of product, and on the other hand differentiation within multi-protein complexes consisting of homologous genes.I will discuss these results in terms of multilevel evolution. in particular in terms of information integration and the alternatives of `individual based´

scholarly journals Monitoring of Pyrethroid Resistance Allele Frequency in the Common Bed Bug (Cimex lectularius) in the Republic of Korea

2020 ◽  
Vol 58 (1) ◽  
pp. 99-102
Author(s):  
Susie Cho ◽  
Heung-Chul Kim ◽  
Sung-Tae Chong ◽  
Terry A. Klein ◽  
Deok Ho Kwon ◽  
...  
Keyword(s):  
Large Scale ◽  
Pyrethroid Resistance ◽  
Single Point ◽  
Point Mutations ◽  
Monitoring Program ◽  
Cimex Lectularius ◽  
Single Point Mutation ◽  
Resistance Allele ◽  
Bed Bugs ◽  
Bed Bug

Two-point mutations (V419L and L925I) on the voltage-sensitive sodium channel of bed bugs (<i>Cimex lectularius</i>) are known to confer pyrethroid resistance. To determine the status of pyrethroid resistance in bed bugs in Korea, resistance allele frequencies of bed bug strains collected from several US military installations in Korea and Mokpo, Jeollanamdo, from 2009-2019 were monitored using a quantitative sequencing. Most bed bugs were determined to have both of the point mutations except a few specimens, collected in 2009, 2012 and 2014, having only a single point mutation (L925I). No susceptible allele was observed in any of the bed bugs examined, suggesting that pyrethroid resistance in bed bug populations in Korea has reached a serious level. Large scale monitoring is required to increase our knowledge on the distribution and prevalence of pyrethroid resistance in bed bug populations in Korea. Based on present study, it is urgent to restrict the use of pyrethroids and to introduce effective alternative insecticides. A nation-wide monitoring program to determine the pyrethroid resistance level in bed bugs and to select alternative insecticides should be implemented.

scholarly journals Transformation of Rickettsia prowazekiito Rifampin Resistance

1998 ◽  
Vol 180 (8) ◽  
pp. 2118-2124 ◽  
Author(s):  
Lyudmila I. Rachek ◽  
Aimee M. Tucker ◽  
Herbert H. Winkler ◽  
David O. Wood
Keyword(s):  
Genetic Manipulation ◽  
Single Point ◽  
Initial Step ◽  
Genetic System ◽  
Single Point Mutation ◽  
Rickettsia Prowazekii ◽  
Obligate Intracellular ◽  
Pcr Products ◽  
Epidemic Typhus ◽  
Obligate Intracellular Pathogen

ABSTRACT Rickettsia prowazekii, the causative agent of epidemic typhus, is an obligate intracellular parasitic bacterium that grows directly within the cytoplasm of the eucaryotic host cell. The absence of techniques for genetic manipulation hampers the study of this organism’s unique biology and pathogenic mechanisms. To establish the feasibility of genetic manipulation in this organism, we identified a specific mutation in the rickettsial rpoB gene that confers resistance to rifampin and used it to demonstrate allelic exchange in R. prowazekii. Comparison of the rpoB sequences from the rifampin-sensitive (Rifs) Madrid E strain and a rifampin-resistant (Rifr) mutant identified a single point mutation that results in an arginine-to-lysine change at position 546 of theR. prowazekii RNA polymerase β subunit. A plasmid containing this mutation and two additional silent mutations created in codons flanking the Lys-546 codon was introduced into the Rifs Madrid E strain of R. prowazekii by electroporation, and in the presence of rifampin, resistant rickettsiae were selected. Transformation, via homologous recombination, was demonstrated by DNA sequencing of PCR products containing the three mutations in the Rifrregion of rickettsial rpoB. This is the first successful demonstration of genetic transformation of Rickettsia prowazekii and represents the initial step in the establishment of a genetic system in this obligate intracellular pathogen.

Simple and Reliable Factor V Genotyping by PNA-Mediated PCR Clamping

1998 ◽  
Vol 79 (04) ◽  
pp. 773-777 ◽  
Author(s):  
Moira Behn ◽  
Marcus Schuermann
Keyword(s):  
Venous Thrombosis ◽  
Large Scale ◽  
Single Point ◽  
Activated Protein C ◽  
Factor V Leiden ◽  
Single Point Mutation ◽  
Factor V ◽  
Apc Resistance ◽  
Novel Approach ◽  
Pcr Rflp

SummaryResistance to activated protein C (APC resistance) is the most common cause of thrombophilia and linked to a single point mutation in the factor V gene (G>A transition at nucleotide 1691). In the past, several PCR based methods have been proposed to determine the allelostatus of individual patients from small amounts of blood DNA including PCR followed by restriction fragment length polymorphism detection (PCR-RFLP), PCR using sequence-specific primers (PCR-SSP) and oligonucleotide ligation assay (OLA). Here, we present a novel approach based on the method of peptide nucleic acid(PNA)-mediated PCR clamping which is extremely sensitive to base pair mismatches. If PNAs specific for the two allelic variants are applied separately in each case a clear discrimination between a heterozygous or homozygous normal or homozygous Factor V Leiden status is possible and no further confirmation step is required. In a prospective study, 60 patients with suspected venous thrombosis events were tested and compared to the conventional PCR-RFLP technique. The concordance between both methods was 100%. PNA-based factor V genotyping, therefore, should be considered for large scale screening of those patients considered to be at risk for deep venous thrombosis.

Production of isomelezitose from sucrose by engineered glucansucrases

Amylase ◽  
2017 ◽  
Vol 1 (1) ◽  
Author(s):  
Gregory L. Côté ◽  
Christopher A. Dunlap ◽  
Karl E. Vermillion ◽  
Christopher D. Skory
Keyword(s):  
Lactic Acid ◽  
Lactic Acid Bacteria ◽  
Biomedical Applications ◽  
Large Scale ◽  
Single Point ◽  
Single Point Mutation ◽  
Scale Production ◽  
Acceptor Substrate ◽  
Large Scale Production ◽  
High Yields

AbstractCertain lactic acid bacteria produce glycosyltransferases known as glucansucrases, which synthesize α-D-glucans via glucosyl transfer from sucrose. We recently reported on the formation of the unusual trisaccharide isomelezitose in low yields by a variety of glucansucrases. Isomelezitose is a rare non-reducing trisaccharide, with the structure α-d-glucopyranosyl- (1→6)-β-d-fructofuranosyl-(2↔1)-α-d-glucopyranoside. In this work, we describe the synthesis of isomelezitose in high yields by variants of glucansucrases engineered to contain a single point mutation at a key leucine residue involved in acceptor substrate binding. Some variants produce isomelezitose in yields up to 57%. This method is amenable to large-scale production of isomelezitose for food, industrial and biomedical applications.

scholarly journals Optimized Cas9 expression improves performance of large-scale CRISPR screening

2021 ◽  
Author(s):  
Joao M. Fernandes Neto ◽  
Katarzyna Jastrzebski ◽  
Cor Lieftink ◽  
Lenno Krenning ◽  
Matheus Dias ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
Functional Genomic ◽  
Editing Efficiency ◽  
Expression Levels ◽  
Genomic Screening ◽  
Invaluable Tool ◽  
Improved Performance

CRISPR technology is an invaluable tool for large-scale functional genomic screening. Genome editing efficiency and timing are important parameters impacting the performance of pooled CRISPR screens. Here we show that by optimizing Cas9 expression levels, the time necessary for gene editing can be reduced contributing to improved performance of CRISPR based screening.

Computational multiscale studies of collagen tissues in the context of brittle bone disease osteogenesis imperfecta

2010 ◽  
Vol 1274 ◽  
Author(s):  
Simone Vesentini ◽  
Alfonso Gautieri ◽  
Alberto Redaelli ◽  
Markus J. Buehler
Keyword(s):  
Osteogenesis Imperfecta ◽  
Large Scale ◽  
Multiple Scales ◽  
Broad Class ◽  
Genetic Disorder ◽  
Single Point ◽  
Collagen Fibrils ◽  
Coarse Grained ◽  
Single Point Mutation ◽  
Length Scales

AbstractOsteogenesis imperfecta (abbreviated as OI) is a genetic disorder in collagen characterized by mechanically weakened tendon, fragile bones, skeletal deformities and in severe cases prenatal death. Even though many studies have attempted to associate specific mutation types with phenotypic severity, the molecular and mesoscale mechanisms by which a single point mutation influences the mechanical behavior of tissues at multiple length-scales remain unknown. Here we review results of a hierarchy of full atomistic and mesoscale simulations that demonstrated that OI mutations severely compromise the mechanical properties of collagenous tissues at multiple scales, from single molecules to collagen fibrils. Notably, mutations that lead to the most severe OI phenotype correlate with the strongest effects, leading to weakened intermolecular adhesion, increased intermolecular spacing, reduced stiffness, as well as a reduced failure strength of collagen fibrils (Gautieri et al., Biophys. J., 2009). Our study explains how single point mutations can control the breakdown of tissue at much larger length-scales, a question of great relevance for a broad class of genetic diseases. Furthermore, by extending the MARTINI coarse-grained force field, we provide a new modeling tool to study collagen molecules and fibrils at much larger scales than accessible to existing full atomistic models, while incorporating key chemical and mechanical features and thereby presents a powerful approach to computational materiomics (Gautieri et al., Journal of Chemical Theory and Computation, 2010). We describe the coarse-graining approach and present preliminary findings based on this model in applying it to large-scale models of molecular assemblies into fibrils.

scholarly journals Dynamics of QoI Sensitivity in Mycosphaerella fijiensis in Costa Rica During 2000 to 2003

2007 ◽  
Vol 97 (11) ◽  
pp. 1451-1457 ◽  
Author(s):  
A. F. Amil ◽  
S. P. Heaney ◽  
C. Stanger ◽  
M. W. Shaw
Keyword(s):  
Costa Rica ◽  
Large Scale ◽  
Spatial Scales ◽  
Quantitative Polymerase Chain Reaction ◽  
Single Point ◽  
Oxidase Inhibitor ◽  
Amplification Refractory Mutation System ◽  
Single Point Mutation ◽  
Mycosphaerella Fijiensis ◽  
Black Sigatoka

From 1997 onward, the strobilurin fungicide azoxystrobin was widely used in the main banana-production zone in Costa Rica against Mycosphaerella fijiensis var. difformis causing black Sigatoka of banana. By 2000, isolates of M. fijiensis with resistance to the quinolene oxidase inhibitor fungicides were common on some farms in the area. The cause was a single point mutation from glycine to alanine in the fungal target protein, cytochrome b gene. An amplification refractory mutation system Scorpion quantitative polymerase chain reaction assay was developed and used to determine the frequency of G143A allele in samples of M. fijiensis. Two hierarchical surveys of spatial variability, in 2001 and 2002, found no significant variation in frequency on spatial scales <10 m. This allowed the frequency of G143A alleles on a farm to be estimated efficiently by averaging single samples taken at two fixed locations. The frequency of G143A allele in bulk samples from 11 farms throughout Costa Rica was determined at 2-month intervals. There was no direct relationship between the number of spray applications and the frequency of G143A on individual farms. Instead, the frequency converged toward regional averages, presumably due to the large-scale mixing of ascospores dispersed by wind. Using trap plants in an area remote from the main producing area, immigration of resistant ascospores was detected as far as 6 km away both with and against the prevailing wind.

ECOGEMS: efficient compression and retrieve of SNP data of 2058 rice accessions with integer sparse matrices

2019 ◽  
Vol 35 (20) ◽  
pp. 4181-4183 ◽  
Author(s):  
Wen Yao ◽  
Fangfang Huang ◽  
Xuehai Zhang ◽  
Jihua Tang
Keyword(s):  
Large Scale ◽  
Sparse Matrices ◽  
Supplementary Information ◽  
Functional Genomic ◽  
Genotype Data ◽  
Graphical Interface ◽  
Cultivated Rice ◽  
Snp Data ◽  
Genomic Studies ◽  
Traditional Approaches

Abstract Summary We proposed to store large-scale genotype data as integer sparse matrices, which consumed much fewer computing resources for storage and analysis than traditional approaches. In addition, the raw genotype data could be readily recovered from integer sparse matrices. Utilizing this approach, we stored the genotype data of 1612 Asian cultivated rice accessions and 446 Asian wild rice accessions across 8 584 244 SNP sites in the ECOGEMS database with 310 MB of disk usage. Graphical interface for visualization, analysis and download of SNP data were implemented in ECOGEMS, which made it a valuable resource for rice functional genomic studies. Availability and implementation The code and data of ECOGEMS are freely available at https://github.com/venyao/ECOGEMS. ECOGEMS is deployed at http://ecogems.ncpgr.cn and http://150.109.59.144: 3838/ECOGEMS/ for online use. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Interrogating Mitochondrial Biology and Disease Using CRISPR/Cas9 Gene Editing

Genes ◽  
2021 ◽  
Vol 12 (10) ◽  
pp. 1604
Author(s):  
Jia Xin Tang ◽  
Angela Pyle ◽  
Robert W. Taylor ◽  
Monika Oláhová
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Gene Editing ◽  
Genetic Manipulation ◽  
Mitochondrial Diseases ◽  
Biological Research ◽  
Genetic Changes ◽  
Mitochondrial Proteome ◽  
A Genome ◽  
Indispensable Tool

Mitochondrial disease originates from genetic changes that impact human bodily functions by disrupting the mitochondrial oxidative phosphorylation system. MitoCarta is a curated and published inventory that sheds light on the mitochondrial proteome, but the function of some mitochondrially-localised proteins remains poorly characterised. Consequently, various gene editing systems have been employed to uncover the involvement of these proteins in mitochondrial biology and disease. CRISPR/Cas9 is an efficient, versatile, and highly accurate genome editing tool that was first introduced over a decade ago and has since become an indispensable tool for targeted genetic manipulation in biological research. The broad spectrum of CRISPR/Cas9 applications serves as an attractive and tractable system to study genes and pathways that are essential for the regulation and maintenance of mitochondrial health. It has opened possibilities of generating reliable cell and animal models of human disease, and with further exploitation of the technology, large-scale genomic screenings have uncovered a wealth of fundamental mechanistic insights. In this review, we describe the applications of CRISPR/Cas9 system as a genome editing tool to uncover new insights into pathomechanisms of mitochondrial diseases and/or biological processes involved in mitochondrial function.

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