scholarly journals Marker Recycling in Candida albicans through CRISPR-Cas9-Induced Marker Excision

mSphere ◽  
2017 ◽  
Vol 2 (2) ◽  
Author(s):  
Manning Y. Huang ◽  
Aaron P. Mitchell
Keyword(s):  
Candida Albicans ◽  
Genetic Manipulation ◽  
Strand Break ◽  
Double Strand Break ◽  
Selection Marker ◽  
Content Type ◽  
Marker Recycling ◽  
Marker Excision ◽  
Selection For ◽  
Selection Markers

ABSTRACT It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology. We describe here a new approach to marker recycling, a controlled sequence of steps in which a genetic marker is selected and then lost. Marker recycling is important for genetic manipulation, because it allows a single selection marker to be used repeatedly. Our approach relies upon the ability of the CRISPR-Cas9 system to make a targeted double-strand break in DNA and the expectation that a double-strand break within a selection marker may promote recombination between directly repeated sequences that flank the marker. We call the approach CRISPR-Cas9-induced marker excision (CRIME). We tested the utility of this approach with the fungal pathogen Candida albicans, which is typically diploid. We used two selection markers, modified to include flanking direct repeats. In a proof-of-principle study, we created successive homozygous deletions in three genes through use of the two markers and had one of the markers available in the final strain for further selection and recycling. This strategy will accelerate the creation of multiple-mutant strains in C. albicans. CRISPR-Cas9 systems have been applied to many organisms, so the genetic design principles described here may be broadly applicable. IMPORTANCE It is critical to be able to alter genes in order to elucidate their functions. These alterations often rely upon markers that allow selection for a rare cell in a population that has incorporated a piece of DNA. The number of alterations that can be accomplished is thus limited by the number of selection markers that are available. This limitation is circumvented by marker recycling strategies, in which a marker is eliminated after its initial use. Then, the marker can be used again. In this report, we describe a new marker recycling strategy that is enabled by recently developed CRISPR-Cas9 technology.

scholarly journals A Double-Strand Break Does Not PromoteNeisseria gonorrhoeaePilin Antigenic Variation

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Lauren L. Prister ◽  
Jing Xu ◽  
H Steven Seifert
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Antigenic Variation ◽  
Dna Structure ◽  
Strand Break ◽  
Double Strand Break ◽  
Content Type ◽  
G4 Dna ◽  
Recombination System ◽  
Guanine Quadruplex ◽  
Pilin Gene

ABSTRACTThe major subunit of the type IV pilus (T4p) ofNeisseria gonorrhoeaeundergoes antigenic variation (AV) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double-strand break (DSB) at the site of thepilEG4 sequence could substitute for G4-directed pilin AV. The G4 motif was replaced by an I-SceI cut site, and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double-strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome, and suppressor mutations that were mainly deletions of the cut site and/or the entirepilEgene accumulated. Thus, thepilEG4 does not act to promote pilin AV by generating a DSB but requires either a different type of break, a nick, or more complex interactions with other factors to stimulate this programmed recombination system.IMPORTANCENeisseria gonorrhoeae, the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface-exposed antigens. This recombination system, known as antigenic variation, uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA; however, much remains unknown about how this structure functions in cells. We show that inducing a break by different means does not allow antigenic variation, indicating that the DNA structure may have a more complicated role.

scholarly journals Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System

mSphere ◽  
2016 ◽  
Vol 1 (3) ◽  
Author(s):  
Kyunghun Min ◽  
Yuichi Ichikawa ◽  
Carol A. Woolford ◽  
Aaron P. Mitchell
Keyword(s):  
Drug Resistance ◽  
Candida Albicans ◽  
Genetic Analysis ◽  
Genome Editing ◽  
Drug Targets ◽  
Gene Deletion ◽  
Genetic Manipulation ◽  
Content Type ◽  
Biological Features ◽  
Link Type

ABSTRACT The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248 ]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

scholarly journals Biased Gene Conversion in Rhizobium etli Is Caused by Preferential Double-Strand Breaks on One of the Recombining Homologs

2015 ◽  
Vol 198 (3) ◽  
pp. 591-599 ◽  
Author(s):  
Fares Osam Yáñez-Cuna ◽  
Mildred Castellanos ◽  
David Romero
Keyword(s):  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Gene Conversion ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Breaks ◽  
Content Type ◽  
Strand Breaks ◽  
Biased Gene Conversion ◽  
Resident Plasmid

ABSTRACTGene conversion, the nonreciprocal transfer of information during homologous recombination, is the main process that maintains identity between members of multigene families. Gene conversion in the nitrogenase (nifH) multigene family ofRhizobium etliwas analyzed by using a two-plasmid system, where each plasmid carried a copy ofnifH. One of thenifHcopies was modified, creating restriction fragment length polymorphisms (RFLPs) spaced along the gene. Once the modified plasmid was introduced intoR. etli, selection was done for cointegration with a resident plasmid lacking the RFLPs. Most of the cointegrate molecules harbor gene conversion events, biased toward a gain of RFLPs. This bias may be explained under the double-strand break repair model by proposing that thenifHgene lacking the RFLPs suffers a DNA double-strand break, so the incoming plasmid functions as a template for repairing the homolog on the resident plasmid. To support this proposal, we cloned an SceI site into thenifHhomolog that had the RFLPs used for scoring gene conversion.In vivoexpression of the meganuclease I-SceI allowed the generation of a double-strand break on this homolog. Upon introduction of this modified plasmid into anR. etlistrain lacking I-SceI, biased gene conversion still favored the retention of markers on the incoming plasmid. In contrast, when the recipient strain ectopically expressed I-SceI, a dramatic reversal in gene conversion bias was seen, favoring the preservation of resident sequences. These results show that biased gene conversion is caused by preferential double-strand breaks on one of the recombining homologs.IMPORTANCEIn this work, we analyzed gene conversion by using a system that entails horizontal gene transfer followed by homologous recombination in the recipient cell. Most gene conversion events are biased toward the acquisition of the incoming sequences, ranging in size from 120 bp to 800 bp. This bias is due to preferential cutting of resident DNA and can be reversed upon introduction of a double-strand break on the incoming sequence. Since conditions used in this work are similar to those in horizontal gene transfer, it provides evidence that, upon transfer, the resident DNA preferentially acquires gene variants.

scholarly journals Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation

2021 ◽  
Author(s):  
Jenny Zhou ◽  
Shu-Ming Li
Keyword(s):  
Cytochrome P450 ◽  
Genetic Manipulation ◽  
Selection Marker ◽  
Cytochrome P450 Monooxygenases ◽  
Cytochrome P450 Enzyme ◽  
Oxidation Reactions ◽  
Functional Identification ◽  
Marker Recycling ◽  
P450 Monooxygenases ◽  
P450 Enzyme

Abstract Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

scholarly journals mSphere of Influence: a Systematic Approach To Dissect Virulence Traits in Candida albicans

mSphere ◽  
2019 ◽  
Vol 4 (3) ◽  
Author(s):  
J. Christian Pérez
Keyword(s):  
Candida Albicans ◽  
Systematic Approach ◽  
Genetic Manipulation ◽  
Homozygous Deletion ◽  
Mammalian Host ◽  
Genetic Screens ◽  
Content Type ◽  
Link Type ◽  
Virulence Traits ◽  
Deletion Library

ABSTRACT J. Christian Pérez studies the interplay between Candida albicans and the mammalian host. In this mSphere of Influence article, he reflects on how “Systematic screens of a Candida albicans homozygous deletion library decouple morphogenetic switching and pathogenicity” (S. M. Noble, S. French, L. A. Kohn, V. Chen, et al., Nat Genet 42:590–598, 2010, https://doi.org/10.1038/ng.605) provided tools and a blueprint for open-ended genetic screens in an organism that had been a challenge for genetic manipulation.

scholarly journals Floxed-Cassette Allelic Exchange Mutagenesis Enables Markerless Gene Deletion inChlamydia trachomatisand Can Reverse Cassette-Induced Polar Effects

2018 ◽  
Vol 200 (24) ◽  
Author(s):  
G. Keb ◽  
R. Hayman ◽  
K. A. Fields
Keyword(s):  
Gene Deletion ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
Selection Marker ◽  
Marker Genes ◽  
Content Type ◽  
Allelic Exchange ◽  
Conditional Expression ◽  
Obligate Intracellular Bacteria ◽  
Green Fluorescent

ABSTRACTAs obligate intracellular bacteria,Chlamydiaspp. have evolved numerous, likely intricate, mechanisms to create and maintain a privileged intracellular niche. Recent progress in elucidating and characterizing these processes has been bolstered by the development of techniques enabling basic genetic tractability. Florescence-reported allelic exchange mutagenesis (FRAEM) couples chromosomal gene deletion with the insertion of a selection cassette encoding antibiotic resistance and green fluorescent protein (GFP). Similar to other bacteria, many chlamydial genes exist within polycistronic operons, raising the possibility of polar effects mediated by insertion cassettes. Indeed, FRAEM-mediated deletion ofChlamydia trachomatistmeAnegatively impacts the expression oftmeB. We have adapted FRAEM technology by employing agfp-blacassette flanked byloxPsites. Conditional expression of Cre recombinase inChlamydiatmeAcontaining a floxed cassette resulted in deletion of the marker and restoration oftmeBexpression.IMPORTANCEC. trachomatisinfections represent a significant burden to human health. The ability to genetically manipulateChlamydiaspp. is overcoming historic confounding barriers that have impeded rapid progress in understanding overall chlamydial pathogenesis. The current state of genetic manipulation inChlamydiaspp. requires further development, including mechanisms to generate markerless gene disruption. We leveraged a stepwise Cre-lox approach to excise selection marker genes from a deleted gene locus. We found this process to be efficient, and the removal of extraneous elements resulted in the reversal of a negative polar effect on a downstream gene. This technique facilitates a more direct assessment of gene function and adds to theChlamydiamolecular toolbox by facilitating the deletion of genes within operons.

scholarly journals Analysis of Repair Mechanisms following an Induced Double-Strand Break Uncovers Recessive Deleterious Alleles in theCandida albicansDiploid Genome

mBio ◽  
2016 ◽  
Vol 7 (5) ◽  
Author(s):  
Adeline Feri ◽  
Raphaël Loll-Krippleber ◽  
Pierre-Henri Commere ◽  
Corinne Maufrais ◽  
Natacha Sertour ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Genome Sequencing ◽  
Strand Break ◽  
Double Strand Break ◽  
Clonal Reproduction ◽  
Strong Increase ◽  
Recessive Lethal ◽  
Independent Events ◽  
Deleterious Alleles ◽  
Strain Sc5314

ABSTRACTThe diploid genome of the yeastCandida albicansis highly plastic, exhibiting frequent loss-of-heterozygosity (LOH) events. To provide a deeper understanding of the mechanisms leading to LOH, we investigated the repair of a unique DNA double-strand break (DSB) in the laboratoryC. albicansSC5314 strain using the I-SceI meganuclease. Upon I-SceI induction, we detected a strong increase in the frequency of LOH events at an I-SceI target locus positioned on chromosome 4 (Chr4), including events spreading from this locus to the proximal telomere. Characterization of the repair events by single nucleotide polymorphism (SNP) typing and whole-genome sequencing revealed a predominance of gene conversions, but we also observed mitotic crossover or break-induced replication events, as well as combinations of independent events. Importantly, progeny that had undergone homozygosis of part or all of Chr4 haplotype B (Chr4B) were inviable. Mining of genome sequencing data for 155C. albicansisolates allowed the identification of a recessive lethal allele in theGPI16gene on Chr4B unique toC. albicansstrain SC5314 which is responsible for this inviability. Additional recessive lethal or deleterious alleles were identified in the genomes of strain SC5314 and two clinical isolates. Our results demonstrate that recessive lethal alleles in the genomes ofC. albicansisolates prevent the occurrence of specific extended LOH events. While these and other recessive lethal and deleterious alleles are likely to accumulate inC. albicansdue to clonal reproduction, their occurrence may in turn promote the maintenance of corresponding nondeleterious alleles and, consequently, heterozygosity in theC. albicansspecies.IMPORTANCERecessive lethal alleles impose significant constraints on the biology of diploid organisms. Using a combination of an I-SceI meganuclease-mediated DNA DSB, a fluorescence-activated cell sorter (FACS)-optimized reporter of LOH, and a compendium of 155 genome sequences, we were able to unmask and identify recessive lethal and deleterious alleles in isolates ofCandida albicans, a diploid yeast and the major fungal pathogen of humans. Accumulation of recessive deleterious mutations upon clonal reproduction ofC. albicanscould contribute to the maintenance of heterozygosity despite the high frequency of LOH events in this species.

scholarly journals Utility of the Clostridial Site-Specific Recombinase TnpX To Clone Toxic-Product-Encoding Genes and Selectively Remove Genomic DNA Fragments

2014 ◽  
Vol 80 (12) ◽  
pp. 3597-3603 ◽  
Author(s):  
Vicki Adams ◽  
Radhika Bantwal ◽  
Lauren Stevenson ◽  
Jackie K. Cheung ◽  
Milena M. Awad ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Genetic Manipulation ◽  
Antibiotic Resistance Genes ◽  
Dna Fragments ◽  
Site Specific ◽  
Content Type ◽  
E Coli ◽  
Marker Recycling ◽  
A Site

ABSTRACTTnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn4451and Tn4453inClostridium perfringensandClostridium difficile, respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes; however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids inEscherichia coliand from marked chromosomalC. perfringensmutants. This methodology enabled the construction of aC. perfringensplc virRdouble mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic toE. coli. We show that TnpX represses expression from its own promoter, PattCI, which can be exploited to facilitate the cloning of recalcitrant genes inE. colifor subsequent expression in the heterologous hostC. perfringens. Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.

scholarly journals Role of DNA Mismatch Repair and Double-Strand Break Repair in Genome Stability and Antifungal Drug Resistance in Candida albicans

2007 ◽  
Vol 6 (12) ◽  
pp. 2194-2205 ◽  
Author(s):  
Melanie Legrand ◽  
Christine L. Chan ◽  
Peter A. Jauert ◽  
David T. Kirkpatrick
Keyword(s):  
Hydrogen Peroxide ◽  
Drug Resistance ◽  
Dna Repair ◽  
Candida Albicans ◽  
Mismatch Repair ◽  
Genome Instability ◽  
Strand Break ◽  
Double Strand Break ◽  
Double Strand Break Repair ◽  
Break Repair

ABSTRACT Drug resistance has become a major problem in the treatment of Candida albicans infections. Genome changes, such as aneuploidy, translocations, loss of heterozygosity, or point mutations, are often observed in clinical isolates that have become resistant to antifungal drugs. To determine whether these types of alterations result when DNA repair pathways are eliminated, we constructed yeast strains bearing deletions in six genes involved in mismatch repair (MSH2 and PMS1) or double-strand break repair (MRE11, RAD50, RAD52, and YKU80). We show that the mre11Δ/mre11Δ, rad50Δ/rad50Δ, and rad52Δ/rad52Δ mutants are slow growing and exhibit a wrinkly colony phenotype and that cultures of these mutants contain abundant elongated pseudohypha-like cells. These same mutants are susceptible to hydrogen peroxide, tetrabutyl hydrogen peroxide, UV radiation, camptothecin, ethylmethane sulfonate, and methylmethane sulfonate. The msh2Δ/msh2Δ, pms1Δ/pms1Δ, and yku80Δ/yku80Δ mutants exhibit none of these phenotypes. We observed an increase in genome instability in mre11Δ/mre11Δ and rad50Δ/rad50Δ mutants by using a GAL1/URA3 marker system to monitor the integrity of chromosome 1. We investigated the acquisition of drug resistance in the DNA repair mutants and found that deletion of mre11Δ/mre11Δ, rad50Δ/rad50Δ, or rad52Δ/rad52Δ leads to an increased susceptibility to fluconazole. Interestingly, we also observed an elevated frequency of appearance of drug-resistant colonies for both msh2Δ/msh2Δ and pms1Δ/pms1Δ (MMR mutants) and rad50Δ/rad50Δ (DSBR mutant). Our data demonstrate that defects in double-strand break repair lead to an increase in genome instability, while drug resistance arises more rapidly in C. albicans strains lacking mismatch repair proteins or proteins central to double-strand break repair.

scholarly journals A TailoredgalKCounterselection System for Efficient Markerless Gene Deletion and Chromosomal Tagging in Magnetospirillum gryphiswaldense

2014 ◽  
Vol 80 (14) ◽  
pp. 4323-4330 ◽  
Author(s):  
Oliver Raschdorf ◽  
Jürgen M. Plitzko ◽  
Dirk Schüler ◽  
Frank D. Müller
Keyword(s):  
Cell Biology ◽  
Protein Targeting ◽  
Genetic Manipulation ◽  
Fluorescent Proteins ◽  
Model Systems ◽  
Content Type ◽  
Magnetospirillum Gryphiswaldense ◽  
Marker Recycling ◽  
Associated Proteins ◽  
Convenient Technique

ABSTRACTMagnetotactic bacteria have emerged as excellent model systems to study bacterial cell biology, biomineralization, vesicle formation, and protein targeting because of their ability to synthesize single-domain magnetite crystals within unique organelles (magnetosomes). However, only few species are amenable to genetic manipulation, and the limited methods for site-specific mutagenesis are tedious and time-consuming. Here, we report the adaptation and application of a fast and convenient technique for markerless chromosomal manipulation ofMagnetospirillum gryphiswaldenseusing a single antibiotic resistance cassette andgalK-based counterselection for marker recycling. We demonstrate the potential of this technique by genomic excision of thephbCABoperon, encoding enzymes for polyhydroxyalkanoate (PHA) synthesis, followed by chromosomal fusion of magnetosome-associated proteins to fluorescent proteins. Because of the absence of interfering PHA particles, these engineered strains are particularly suitable for microscopic analyses of cell biology and magnetosome biosynthesis.

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