Targeted Nucleotide Exchange in Saccharomyces cerevisiae Directed by Short Oligonucleotides Containing Locked Nucleic Acids

Parekh-Olmedo, Hetal; Drury, Miya; Kmiec, Eric B.

doi:10.1016/s1074-5521(02)00236-3

Cited by 51 publications

(44 citation statements)

References 30 publications

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“…This assay has previously enabled an investigation of optimal targeting vectors and proteins essential for the pairing phase of the TNE reaction (Liu et al, 2001;Liu et al, 2002a;Liu et al, 2002b;Liu et al, 2003;Liu et al, 2004;Parekh-Olmedo et al, 2002). Studies have shown that an MSSO is capable of directing the repair of a chromosomal insertion mutation, but the repair of a chromosomal deletion mutation was not investigated.…”

Section: Modified Single-stranded Oligonucleotides Direct the Repair mentioning

confidence: 99%

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

Maguire

Kmiec

2007

Gene

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The mechanism by which modified single-stranded oligonucleotides (MSSOs) direct base changes in genes is not completely understood, but there is evidence that DNA damage, repair and cell cycle checkpoint proteins are involved in the targeted nucleotide exchange (TNE) process. We are interested in the role of the mismatch repair protein, Msh2 in the repair of a frameshift mutation in both yeast and mammalian cells. We show that this protein exerts different and opposing influences on the TNE reaction in MSH2 deficient yeast compared to MSH2 -/-mammalian cells and in wildtype cells that have RNAi silenced Msh2. Data from yeast show a 10 fold decrease in the targeting frequency whereas mammalian cells have an elevated correction frequency. These results show that in yeast this protein is required for efficient targeting and may play a role in mismatch recognition and repair. In mammalian cells, Msh2 plays a suppressive role in TNE reaction by either precluding the oligonucleotide annealing to the target gene or by maintenance of a cell cycle checkpoint induced by the MSSO itself. These results reveal that the mechanism of TNE between yeast and mammalian cells is not conserved, and demonstrate that the suppression of the TNE reaction can be bypassed using RNAi against MSH2 designed to knockdown its expression.

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Section: Modified Single-stranded Oligonucleotides Direct the Repair mentioning

confidence: 99%

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

Maguire

Kmiec

2007

Gene

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“…Hyg3S/74NT is a 74-mer that is specific for binding to the nontranscribed strand and contains three terminal phosphorothioate linkages. 8,11,12 Also shown is the target sequence of the mutant, which contains a TAG stop codon. Figure 2c illustrates the structure of the b-globin YAC and nucleotides targeted for editing are specified.…”

Section: Resultsmentioning

confidence: 99%

“…In particular, BAC modifications must be conducted in a specially engineered strain, while our method can be used in essentially any genetic background, targeting either a plasmid or a chromosome. 8,11,12 More importantly, BACs have a restricted length of foreign DNA (p300 kb) capable of being stably maintained, while YACs can hold up to 2 Mb (see, for review, Huxley 19 ). Therefore, YACs enable targeting of introns and regulatory as well as coding regions, and could reveal interactions between genes linked too loosely to be present on a single BAC or PAC.…”

Section: Aj Van Brabant Et Almentioning

confidence: 99%

“…(c) Schematic of the YAC containing the human b-globin locus and the bThal1 and bThal2 sequences that were changed by the corresponding MSSOs. The YAC contains approximately 230 kb of genomic DNA from human chromosome, 11 indicated by the shaded region. The unshaded regions represent the yeast sequences that are on either end of the YAC (not drawn to scale).…”

Section: Introductionmentioning

confidence: 99%

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Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting

Brabant¹,

Williams²,

Parekh-Olmedo

et al. 2004

Pharmacogenomics J

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A single-nucleotide polymorphism (SNP) in a human gene can alter the behavior of the corresponding protein, and thereby affect an individual's response to drug therapy. Here, we describe a novel dual-targeting approach for introducing an SNP of choice into virtually any gene, through the use of modified single-stranded oligonucleotides (MSSOs). We use this strategy to create SNPs in a human gene contained in a yeast artificial chromosome (YAC). In the dual-targeting protocol, two different MSSOs are designed to edit two different bases in the same cell. A change in one of these genes is selective while the other is non-selective. We show that the population identified by selective pressure is enriched for cells that bear an edited base at the nonselective site. YACs with human genomic inserts containing particular SNPs or haplotypes can be used for pharmacogenomic applications, in cell lines and in transgenic animals.

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“…MSOs that are 70 to 80 bases long appear to be optimal for TNE on both episomes and chromosomes (12). Alternative modifications, such as locked nucleic acid residues, can also protect against nuclease digestion while high levels of activity are maintained (20). Locked nucleic acid residues may eventually prove to be useful since they have low levels of cellular toxicity, especially in mammalian cells (27).…”

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confidence: 99%

Nuclease Activity of Saccharomyces cerevisiae Mre11 Functions in Targeted Nucleotide Alteration

Liu

Usher

et al. 2003

Appl Environ Microbiol

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Oligonucleotides can be used to direct site-specific changes in genomic DNA through a process in which mismatched base pairs in the oligonucleotide and the target DNA are created. The mechanism by which these complexes are developed and resolved is being studied by using Saccharomyces cerevisiae as a model system. Genetic analyses have revealed that in all likelihood the reaction occurs in two phases: DNA pairing and DNA repair. While the former phase involves strand assimilation, the latter phase likely involves an endonucleolytic processing step that leads to joint resolution. In this study, we established the importance of a functioning MRE11 gene in the overall reaction, as yeast strains deficient in MRE11 exhibited severely reduced activity. The activity could be rescued by complementation with wild-type MRE11 genes but not with MRE11 alleles lacking the nuclease function. Taken together, the data suggest that Mre11 provides nuclease activity for targeted nucleotide exchange, a process that could be used to reengineer yeast genes.Genetic reengineering in lower eukaryotes holds promise for the creation of host strains that can produce useful metabolites or express desirable phenotypes. The majority of efforts in yeasts have focused on the development of knock-out strategies, where the aim is to completely disable the gene but in many cases the most desirable outcome is a (subtle) modulation of gene function. Thus, new approaches that are simple and straightforward and do not require multiple cloning steps must be developed to meet this goal. A direct approach centers around alteration of single bases at specific sites in chromosomal genes without the need for integration or extensive genetic rearrangement.Single-stranded DNA molecules can be used to direct the alteration or exchange of specific nucleotides in the genome of Saccharomyces cerevisiae. Pioneering work by Moerschell and colleagues (16) demonstrated the feasibility of base repair in the CYC1 gene, a gene encoding cytochrome c isoform 1. A variety of point and frameshift mutations within CYC1 were reversed by the transformation of single strands of DNA in approximately 0.0003 to 0.002% of the cells (28). The mechanism of conversion, however, was not fully elucidated in these studies, although the reaction was found to be dose dependent and exhibited an interesting strand bias. In a similar line of experiments, we have begun to examine the potential for using modified single-stranded DNA oligonucleotides (MSOs) to repair point and frameshift mutations in the yeast chromosome, and our long-term goal is to apply the technique to mammalian cells (22; see reference 2 for a review). To this end, we focused on examining the mechanism by which MSOs direct nucleotide alteration in yeast cells.

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Targeted Nucleotide Exchange in Saccharomyces cerevisiae Directed by Short Oligonucleotides Containing Locked Nucleic Acids

Cited by 51 publications

References 30 publications

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

Multiple roles for MSH2 in the repair of a deletion mutation directed by modified single-stranded oligonucleotides

Gene editing of a human gene in yeast artificial chromosomes using modified single-stranded DNA and dual targeting

Nuclease Activity of Saccharomyces cerevisiae Mre11 Functions in Targeted Nucleotide Alteration

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