Allelic replacement strategy

tuberculosis transposition mutant libraries and is efficient for gene exchange mutagenesis. We hypothesized that an additional May 23, 2008 · This strategy allows the recycling and reuse of the same resistance marker for another round of allelic replacement. gene Our strategy can be easily adopted for replacing any existing genes with a desired allele in rice. By repeatedly introducing a pool of targeting oligos into a cell and incorporating bacteriophage lambda red recombinase, MAGE enables many modifications at different chromosomal locations simultaneously and Oct 23, 2010 · In F. Allelic exchange is the most versatile means of genetic manipulation of bacterial strains. author: Wang, Harris H. Discover the world's research 25+ million members allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. Subculture twice at 25 °C was critical to promote an allelic exchange rate in S. To ensure sufficient DRT for HDR, we delivered both the plasmid and the free DRT into 291 rice (Japonica cv. Zhonghua 11) calli by particle www. Upon a more focused in vitro analysis of EB entry examined 1 h postinfection, the tarP mutant demonstrated a significant reduction in invasion compared to that of wild-type C May 26, 2024 · The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. May 23, 2011 · The efficiency of this allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. tularensis contained the following elements: (i) an antibiotic marker, the chloramphenicol resistance gene (crg) from the Shigella flexneri plasmid pSa [19], which confers chloramphenicol resistance in F. In addition, by eliminating the cloning requirement for construction, we were able to reliably assemble all of the mutation constructs Apr 20, 2020 · Regardless, our newly demonstrated ability to use allelic replacement for cis-complementation should provide an effective means of complementation for future studies. G. Gene conversion can be either allelic, meaning that one allele of the same gene replaces another allele, or ectopic, meaning that one paralogous DNA sequence converts another. This unit describes methods developed in our laboratory for gene disruption by allelic replacement and transposition. Two colony color-types resulted from this allelic replacement strategy, one type was pink and the other was indistinguishable from the WT parental strain. D. By this method a segment of DNA within a bacterial chromosome or episome can be replaced with a Feb 6, 2014 · While genetically tractable, the manipulation of S. Feb 2, 2014 · The pKOR1 plasmid used for allelic replacement in our laboratory is a modified version of pTS1 that encodes the antisense secY RNA for counter-selection during allelic exchange and the lambda recombination cassette (Gateway Technology, Invitrogen) that permits rapid cloning of mutant alleles without the use of restriction enzymes and ligases Aug 1, 2008 · Abstract. Ellermeier, 2019, J Bacteriol 201:e00711-18 For allelic replacement mutagenesis, two-step PCR is performed . We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5′ and 3 A groundbreaking strategy aimed at streamlining the process for marker-less allelic replacement in Streptococcus pneumoniae, a Gram-positive bacterium and leading cause of pneumonia, meningitis, and ear infections is introduced. Modified Bases Enable High-Efficiency Oligonucleotide-Mediated Allelic Replacement via Mismatch Repair Evasion Feb 12, 2008 · Gene replacement via homologous recombination is a fundamental tool for the analysis of gene function. Oct 22, 2015 · To facilitate allelic replacement, an allelic exchange vector is engineered with a copy of the gene of interest, which contains the desired mutation, as well as flanking regions of DNA that are Sep 30, 1997 · A system enabling the positive selection of insertional mutants having lost the delivery vector was developed, which allowed the construction of M. They focused on an insect protein known as the voltage-gated sodium channel (VGSC) which is a target for a widely used class of insecticides. Expand Modified bases enable high-efficiency oligonucleotide-mediated allelic replacement via mismatch repair evasion. tularensis and other organisms, where the pC194 replicon can replicate, this vector can be used to deliver the I-SceI for allelic replacement strategies. 2015), with the corresponding elite allele in indica rice, using CRISPR/Cas9-mediated gene-editing technol-ogy. Among the 602 genes analyzed, we found that 326 genes (54%) displayed a significant difference (at least a twofold difference; for details, see Methods) in at least one individual. Site-specific recombination systems are capable of eliminating such markers Download scientific diagram | Outline of the vector-free allelic replacement mutagenesis strategy as originally described by Chalker et al. We executed this 2-stage allele swap May 1, 2004 · The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. Müh, A. After the construction of this suicide delivery vector, allelic exchange involves two steps (Fig. g. See Full PDF Download PDF. Northern blot analysis provided proof that the ssp genes are transcribed as an operon and that inactivation of sspA was achieved without affecting transcription of downstream genes (Fig. , 2007; Winkler and Kao, 2012; Sahu et al. net bombardment (Figure 1C). Plasmid transformation of staphylococci and growth at 43 °C, a non-permissive condition for A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. (B) Southern blot analysis. Apr 1, 2009 · Moreover, this strategy requires the analysis of a far greater number of clones to identify those with double allelic replacement. Zhonghua 11) calli by particle bombardment (Figure 1C). difficile (U. This single modification We tested our strategy of precise allelic replacement without additional selection pressures in rice (Figure 1C). However, global MMR inactiva- Jul 15, 2011 · The average allelic replacement frequency observed was 37 ± 19% after 18 cycles, and 42% of the population was unconverted after 18 cycles; we observed 1 to 10 converted alleles per clone across the remaining population . Squares indicate codons. tularensis [10]; (ii) an origin of transfer (mob) and (iii) the SacB gene Feb 16, 2016 · Allelic replacement frequencies for all three strains were measured at each locus either by colorimetric assay or allele-specific PCR, as described previously . aureus shuttle vector, permits rapid cloning via lambda recombination and ccdB selection. dc. Allelic replacement in the Burkholderia genus has been problematic due to the lack of appropriate counter-selectable and selectable markers. aureus in 5 days, from the start of cloning to isolation of genomic DNA for confirmatory whole-genome sequencing. The conventional Janus protocol involves two transformation steps using multiple PCR-assembled products containing the Janus cassette May 23, 2011 · This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. 1B. The ability to genetically manipulate bacteria is a staple of modern molecular microbiology. pf, perfect match; 1 mm, 1-bp mismatch. Pannullo, D. Menu. Nov 22, 2023 · The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. An allelic-exchange system that allows for the replacement of the transposon with useful genetic markers and fluorescent reporter genes is generated and provides an unparalleled resource for the study of S. contributor Dec 16, 2023 · Gene knockout is a widely used technique for engineering bacterial genomes, investigating the roles of genes in metabolism, and conferring biological characteristics. Using this method, we modified 2 base pairs in a gene of interest without altering the final sequence of the CRISPR cut sites. The result of this allelic replacement strategy is restoration of the WT gene (left), or deletion of the pigP gene (right). aureus, adapted from an rpsL-based counterselection strategy (Ortiz-Martin, 2006, Russell, Dahlquist, 1989, Sander, 1995, Skorupski, Taylor, 1996). 2 ). Plasmid pKOR1, an Escherichia coli/S. A better understanding of Mycobacterium tuberculosis virulence mechanisms is highly dependent on the design of efficient mutagenesis systems. plasmids • Aerobactin, a siderophore encoded by Allelic replacement with pDG3 using tse2 as a counter D. aureus rocA were generated at frequencies that obviated the need for anti- Allelic replacement has been used extensively to Schematic diagram of allele replacement strategy using vector pKO5. , mFRT May 26, 2024 · innovative strategy to achieve marker-less allelic replacement through a single transformation step. In multiply modified strains, the number of mutated genes usually correlates with the number of selection markers in the strain's chromosome. If the wFRT cassette is used in the first round of gene replacement, then the resistance marker in the second round of allelic replacement should be flanked identically by other FRTs with different spacer sequences (e. This work develops a novel allelic replacement vector, merging physical standardization concepts and a screen-ing procedure based on a simple colorimetric assay, never applied before in marker-less allelic replacement methods for E. reuteri ΔArAT mutant was generated by an in-frame deletion strategy using the allelic replacement vector pJAM083 as described previously 17,72. Genome engineering using single-stranded oligonucleotides is an efficient method for generating Jan 2, 2017 · To address these issues, we developed a 2-stage, marker-assisted strategy to facilitate precise, "scarless" edits in Drosophila with a minimal requirement for molecular screening. Plasmid transformation of staphylococci and growth at 43 degrees C, a non-permissive condition for May 23, 2011 · https://orcid. Transfected parasite DNA (X/P digested) shows the uninterrupted endogenous gene (open arrow) and episomal plasmid (gray arrow), but no evidence Apr 10, 2023 · a, Schematic of precise HDR for mono-allelic G>A replacement in WT/WT mESCs. , based on a traditional allelic exchange strategy. 1). Important principles applicable to any gene-targeting experiment should be considered when discussing gene-targeting techniques. However, global MMR inactivation generally results in Mar 16, 2020 · An allelic replacement approach for simultaneous disruption and complementation of the pfpkg gene. The counter-selectable marker sacB, commonly Consequently, it was necessary to inactivate sspA in S. Salmonella. Here, we developed a two-end selection marker (Two-ESM) method for Genetic Tools To Enhance the Study of Gene Function and Regulation in Staphylococcus aureus. Herein, we developed a rapid, efficient, and simple method for the knockout of long gene cassettes in Pseudomonas spp. (Fig. Tùng Hoàng. This single modification allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. Selected clones carrying the desired modifications were verified by capillary sequencing, after which the MAGE cycles targeting a new locus were continued. F. DNA sequences flanking the target gene ( ϳ 500 bp Gene conversion is the process by which one DNA sequence replaces a homologous sequence such that the sequences become identical after the conversion event. With this optimized approach, a site-specific mutation can be introduced into S. Expand. We have previously observed similar sequence context effects on the allelic-replacement efficiency at the target site due to inhibitory hairpin secondary structures of the oligo ( 9 ). Our approach involves the integration of an additional gene downstream region upstream of the Janus cassette, resulting in a modified genetic arrangement. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens including in vivo studies without the need for A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. However, it is challenging in mutating the essential genes with the traditional method by using a selection marker, since the first step of essential gene knockout will result in a lethal phenotype. org. We could not obtain amplicons of cpsJ Download scientific diagram | Strategy for allelic replacement by a suicide vector. After allelic replacement, loss of the I-SceI delivery plasmid is desired, so an easy strategy to identify bacteria that have lost this plasmid would be to grow cultures without Jun 1, 2005 · Overall strategy for allelic replacement mutagenesis and gene knockout by homologous recombination. Restriction sites: X, XmaI; N, NsiI; S, SphI; P, PacI. We also provide a protocol for bacteriophage-mediated transduction of mutants marked with selectable alleles and describe plasmid utilization for VOL. . aureus remains challenging. About Europe PMC; Preprints in Europe PMC Using this strategy, allelic replacements in S. We thank Olivier Poupel for invaluable assistance with graphic representations, Fadhila Rayah for assistance with the early construction stages of pMAD Jul 16, 2019 · Subsequent methods to address the function of a specific gene have been done by targeted mutagenesis using either a Group-II intron based strategy to program insertions (see TargeTron, Chap. However, global MMR inactiva- Efficient allelic replacement in rice by gene editing: a case study of the NRT1. In this Journal of Bacteriology issue, Müh et al extend the genetics toolbox by developing a CRISPRi strategy for gene silencing in C. 2). , Serratia marcescens ), conjugation constitutes an alternative approach to introduce the plasmid DNA editing substrates ( Snyder Jun 24, 2014 · Allelic replacement was performed using the Janus cassette (P-kan-rpsL 1 ) or the Sweet Janus cassette (P-sacB-kan-rpsL 1 ) as described previously (38, 60). We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of Nov 2, 2011 · For this approach, we incorporated the pheS* open reading frame into a hybrid positive- and negative-selection cassette, which facilitated the allelic replacement strategy we used to introduce the constructs. However, global MMR inactiva-tion generally results in significant accumulation of undesired background mutations. Jan 7, 2021 · Here, we introduce a streamlined allelic exchange protocol using IMxxB Escherichia coli and the plasmid pIMAY-Z. Plasmid pKOR1, an Escherichia coli / S. Spontaneous revertants of Janus can lead to high level of false-positives during negative selection, which necessitate a time-consuming post-selection screening process. To facilitate allelic replacement, an allelic exchange vector is engineered with a copy of the gene of interest, which contains the desired mutation, as well as flanking regions of DNA that are homologous to the recipient chromosome. aureus by allelic replacement without the use of a counter-selection marker. coli genome [23]. Since the 2000’s, marker-less mutants of Streptococcus pneumoniae (Spn Resulting strains were then subjected to allelic-replacement mutagenesis of the native allele under inducing conditions for expression of the ectopic copy of the gene. S. May 1, 2008 · Strategy for replacing the wild-type PaAnt1 + allele by the mutated PaAnt1 296 allele. (A) Features of a typical allele replacement vector include a positive selection marker for antibiotic resistance marker (zeo May 1, 2015 · Here, we describe an additional system for allelic replacement in S. Golden Gate cloning is a molecular cloning strategy allowing researchers to simultaneously and directionally assemble multiple DNA fragments to construct target plasmids using type IIs restriction enzymes and T4 DNA ligase in the same reaction system. May 24, 2024 · innovative strategy to achieve marker-less allelic replacement through a single transformation step. Precise replacement of an existing allele in commercial cultivars with an elite allele is a major goal in crop breeding. Weiss, and C. In the second PCR reaction, the up- and downstream fragments are fused to the amplified Kan R gene using primer L-F and R-R. 7 of this volume) or by allelic replacement using an R6K-based suicide plasmid, pKNG101, that lacks a functional pir gene, encoding the key replication Jul 15, 1998 · We have devised a novel allelic replacement strategy to introduce mutant pppk‐dhps genes at the endogenous locus, retaining transcriptional control from the autologous promoter. Pointers indicate DSB sites. We show here that deletion of the PaKu70 gene is an efficient strategy for improving gene manipulation in this organism. Using an allelic replacement strategy with a nonpolar cassette, we inactivated the response regulator of four TCSs that have only weak homology with TCS genes of known or inferred function in other bacteria. Two pairs of gene-specific primers, L-F/L-R and R-F/R-R, are used to amplify the left and right flanking regions of each target gene, generating PCR products of 500 to 800 bp in length. 1B allele with the Feb 11, 2020 · Abstract Targeted gene mutation by allelic replacement is important for functional genomic analysis and metabolic engineering. (1) 600 bp fragments 5 and 3 to the PaAnt1 gene were ampliWed by PCR using the primer pairs (Ant1-5/Ant1-5r Aug 26, 2012 · (a) Schematic of the pfcrt allelic replacement strategy. 1B gene, which encodes a nitrate transporter (Hu et al. Here, we present a novel strategy using oligos containing chemically modified bases (20-Fluoro-Uridine, Dec 17, 2006 · Recombineering frequency can therefore be expressed as the number of allelic exchange recombinants per microgram DNA divided by the cell competency (that is, the number of plasmid DNA Genetic Tools for Allelic Replacement in Burkholderia Species. 1B gene between japonica and indica rice is responsible for the improved nitrogen use efficiency in indica rice. Step 1 , the entire plasmid is integrated into the chromosome by a single-crossover between the homologous genes Dec 28, 2007 · Allelic replacement in staphylococci is frequently aided by antibiotic resistance markers that replace the gene (s) of interest. The two different merodiploid resolution strategies enabled by pEXKm5 are illustrated. Streptomycin belongs to the aminoglycoside class of antibiotics that inhibit prokaryotic protein synthesis by binding to the Blue-white screening strategy can be used for mark-less gene deletion in . May 28, 2003 · A suicide vector designed to be used for allelic replacement in F. (6). This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. E. aureus rocA were generated at frequencies that obviated the need for antibiotic marker selection. Jul 26, 2005 · A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Hygromycin-resistant calli were transferred onto regeneration media. Show simple item record. Aug 5, 2016 · To overcome this challenge, I devised an allele-replacement strategy that implements a visible, selectable marker and is designed to facilitate the insertion of a series of alleles without the Version of Record. Europe PMC. pneumoniae R6 with those of Bacillus subtilis, Enterococcus faecalis, Escherichia coli, and Staphylococcus aureus and selected 693 candidate target genes with > 40% amino acid sequence identity to the corresponding genes in at least two of the other species. Since the 2000's, marker-less mutants of Streptococcus pneumoniae (Spn) have been made by allelic-exchange predominantly using the kanR-rpsLcassette known as "Janus". To find potential targets of novel antimicrobial agents, we identified essential genes of Flexible Strategy and Social Evolution MARY JANE WEST-EBERHARD* Smithsonian Tropical Research Institute, Balboa, Panama One way in which a new organismic character can originate is via allelic replacement: a mutant genetic allele increases in frequency under selection favouring the affected phenotype(s) until it replaces other, alterna- Nov 10, 2021 · The commonly used methods for gene deletion in Burkholderia are based on disruption of the targeted genes with an antibiotic-resistance marker, through an integrative vector or by allelic replacement of the target gene with an antibiotic resistance cassette. Our strategy can be easily adopted for replacing any existing genes with a desired allele in rice. aureus RN6390 using a nonpolar allelic replacement strategy. In addition, the two recombined alleles cannot be distinguished by virtue of different resistance marker genes, thus making a stringent verification of correct marker gene integration more demanding. Using an allelic replacement strategy to insertionally inactivate the wbbO gene from a virulent strain of K. These measurements suggest the evolution of two types of cells in our mixed cultures: one that appears largely resistant to Oct 1, 2011 · We report a simple technique using pKFT to construct a markerless gene deletion mutant in S. The pZFN crt - bsd plasmid encodes pfcrt -specific ZFNs, with expression driven by the calmodulin promoter ( cam ). (i) Double cross-over. A single nucleotide polymorphism in the NRT1. Moreover, 170 genes (28%) showed a difference greater than fourfold. To ensure sufficient DRT for HDR, we delivered both the plasmid and the free DRT into 291 rice (Japonica cv. Apr 17, 2023 · L. Feb 13, 2016 · Since the development of standard genetic tools is one of the hallmarks of synthetic biology, strongly facilitating and speeding up the recombinant strain construction process [ 17, 18 ], we designed a vector that is compatible with commonly used BioBrick™ standards (RFC10, RFC12 and RFC23) [ 19 ]. aureus. This technique is very simple and useful to facilitate genetic research on S. coli, that can be virtually used with any growth medium and host. We further show that the mismatched bases between the full length oligo and the chromosome are often not incorporated at the target site, probably due to nuclease activity at the 5' and 3' termini of Jun 24, 2014 · The Janus cassette permits marker-free allelic replacement or knockout in streptomycin-resistant Streptococcus pneumoniae (pneumococcus) through sequential positive and negative selection. jipb. Jan 14, 2022 · "Through these allelic replacement strategies, it should be possible to achieve the same degree of pest control with far less application of insecticides," said Bier. About. This study compared the genome of S. However, global MMR inactiva- May 23, 2011 · This strategy increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. Although the 3′ non‐coding region of the pppk‐dhps gene is replaced with the 3′ end of hsp86 in these constructs ( Figure 3C ), this appears not to affect RNA In F. After allelic replacement, loss of the I-SceI delivery plasmid is desired, so an easy strategy to identify bacteria that have lost this plasmid would be to grow cultures without Nov 18, 2002 · Differential Allelic Gene Expression Is Common in the Human Genome. Of note, in difficult-to-transform bacterial strains (e. Streptomycin belongs to the aminoglycoside class of antibiotics that inhibit prokaryotic protein synthesis by binding to the Aug 21, 2009 · This step results in integration of the allelic replacement construct into the chromosome by homologous recombination between cloned and chromosomal sequences and can be visualized by the appearance of blue colonies on Km- and X-Gluc-containing medium. However, this event is rare in organisms like the filamentous fungus Podospora anserina. This strategy produced test strains whereby cellular viability was uniquely dependent on the presence of inducer and provided a direct and absolute confirmation of genetic May 23, 2011 · A novel strategy using oligos containing chemically modified bases in place of the standard T, C, A or G to avoid mismatch detection and repair is presented, which increases transient allelic-replacement efficiencies by up to 20-fold, while maintaining a 100-fold lower background mutation level. Nowadays, the most used method for gene inactivation is based on two recombination Allelic variations can be generated through gene targeting by the introduction of mutations such as amino acid substrtutions, insertions and deletions, alterations in regulatory elements, and complete gene replacement. , 2013). May 23, 2011 · However, this improved allelic-replacement efficiency was not seen with other mismatch pairs, suggesting that such a strategy might be dependent on sequence context. 2008, Applied and Environmental Microbiology. Apr 10, 2018 · To investigate gene function in Bacillus anthracis, a high-efficiency cloning system is required with an increased rate of allelic exchange. pneumoniae O1:K2, we examined host response to wild-type and O-antigen-deficient bacteria in terms of complement binding, susceptibility to serum and whole-blood bactericidal activity, host lethality, and pulmonary inflammatory response Feb 3, 2009 · Primers used for PCR/restriction in the allelic replacement strategy are indicated (semiarrows). Here, we replaced the japonica rice NRT1. contributor. In the first PCR reaction, the aphIII gene (Kan R) and the up- and downstream regions of target genes are amplified. allelic replacement strategy is highly dependent on avoidance of the endogenous mismatch repair (MMR) machinery. May 23, 2011 · Here, we presented an alternative strategy that uses oligonucleotides containing modified bases 2′-Fluoro-deoxyUridine, 5-Methyl-deoxyCytidine, 2,6-Diaminopurine or Iso-deoxyGuanosine to effectively evade MMR to improve allelic-replacement efficiency in a strain with native MMR activity. Briefly, A 1,407 bp PCR product was amplified Jun 21, 2019 · Clostridioides difficile genetics has rapidly advanced in recent years thanks to the development of tools for allelic replacement and transposon mutagenesis. By using the ΔPaKu70 strain, it is now possible (1) to produce deletion mutants May 1, 2015 · Here, we describe an additional system for allelic replacement in S. Having established the pfpkg_2lox parasite line, we next further genetically modified the parasites to enable simultaneous disruption and replacement of the pfpkg gene, using the paired, mutually incompatible lox sites within the 2loxPint intron. This novel plasmid for allelic replacement Feb 1, 2006 · Using this strategy, allelic replacements in S. Herein, we precisely replaced the japonica NRT1. Here, we present a novel strategy using oligos containing chemically modified bases (20-Fluoro-Uridine, May 14, 1999 · Gene Therapy - Allele replacement: an application that permits rapid manipulation of herpes simplex virus type 1 genomes This strategy is useful as BAC sequences may be problematic if the Mar 25, 2018 · We tested our strategy of precise allelic replacement without additional selection pressures in rice (Figure 1C). A system Mar 1, 2024 · MAGE introduces precise edits at multiple sites scarlessly through oligonucleotide-mediated allelic replacement in E. The upstream and downstream sequences of the target gene Sep 10, 2020 · Besides allelic replacement, gene insertions and deletions can be generated using this strategy (Lee et al. 70, 2004 ALLELIC REPLACEMENT IN GRAM-POSITIVE BACTERIA We thank Tarek Msadek, Georges Rapoport, and Patrick TrieuCuot for many helpful discussions and critical reading of the manuscript. Jan 1, 2006 · A method for rapid selection of allelic replacement mutations in the chromosome of Staphylococcus aureus is described. Jan 18, 2022 · In the new study, the researchers used this “allelic drive” strategy to restore genetic susceptibility to insecticides, similar to insects in the wild prior to their having developed resistance. The mutant strains were analyzed by expression microarray analysis at four time points and tested in two mouse infection models. um vx ly aj qw hk qc wn yo ay