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Delitto perfetto
Delitto perfetto is a genetic technique for in vivo site-directed mutagenesis in yeast. This name is an Italian term for perfect deletion and is also an idiom for perfect murder. The name refers to the ability of the technique to create desired genetic changes without leaving any foreign DNA in the genome.
Background
This technique was developed by a group at the National Institute of Environmental Health Sciences (NIEHS) composed of Michael A. Resnick, Francesca Storici (now at Georgia Institute of Technology), and L. Kevin Lewis (now at Southwest Texas State University). The method uses synthetic oligonucleotides in combination with the cellular process of homologous recombination. Consequently, it is well suited for genetic manipulation of yeast, which has highly efficient homologous recombination. The delitto perfetto approach has been used to produce single and multiple point mutations, gene truncations or insertions, and whole gene deletions (including essential genes).
Advantages
The primary advantage of this technique is its ability to eliminate any foreign DNA from the genome after the mutagenesis process. This ensures there are no selectable markers or exogenous sequences used for targeting left in the genome that may cause unforeseen effects. The delitto perfetto technique is also simpler compared to other methods for in vivo site-directed mutagenesis. Other methods require multiple cloning steps and extensive DNA sequencing to confirm mutagenesis, which is often a complicated and inefficient process.[1][2][3] There is great flexibility in this approach because after the CORE cassette is inserted (see Method Overview for details), multiple mutations in the gene of interest can be made easily and quickly. This method can be applied to other organisms where homologous recombination is efficient, such as the moss Physcomitrella patens, DT40 chicken cells, or E. coli. analysis In addition, human genes can be studied and similarly genetically manipulated in yeast by using yeast artificial chromosomes (YACs).
Disadvantages
Since the delitto perfetto technique is based on homologous recombination, this process must be functional in the cells for the technique to work. In Saccharomyces cerevisiae, the RAD52 gene is essential for homologous recombination, and thus is required for the delitto perfetto method. The method is useful only for applications where selectable markers are not necessary. For example, mutagenized yeast strains cannot be used for further genetic analysis such as tetrad analysis. Markers would have to be inserted into the appropriate locus in a separate process.
Delitto perfetto
Technical drawbacks
Cost of oligonucleotides Mutagenesis is limited to region of genome surrounding the inserted cassette Limited number of reporter genes (restricted by available CORE cassettes) Low efficiency for certain applications (e.g. deleting essential genes)
Method Overview
Delitto Perfetto is a two step method for in vivo mutagenesis. In the initial step, the CORE cassette is inserted in the region of interest by homologous recombination. Subsequently, the CORE cassette is replaced with DNA containing the mutation of interest.
CORE cassettes
The CORE cassette contains both a COunterselectable marker and REporter gene. The reporter gene allows for the selection of yeast cells that receive the CORE cassette during the first step of the process. The counterselectable marker allows for the selection of yeast cells that lose the CORE cassette by the integration of the mutated oligonucleotide during the second step of the process. There are a variety of CORE cassettes to choose from, which contain a variety of reporter genes, counterselectable makers and additional features.[4]
Reporter genes
kanMX4 - allows for growth in media containing Geneticin hyg - allows for growth in media containing Hygromycin B.
Counterselectable markers
KlURA3 - prevents growth in media containing 5-flouroorotic acid. GAL1/10-p53 prevents growth in media containing galactose. It encodes a toxic mutant of p53 under a GAL1 promoter.[5]
Delitto perfetto
Additional features
GAL1-I-SceI Increases the efficiency of targeting to the CORE cassette-containing chromosome in diploid cells. It contains the restriction endonuclease SceI under the GAL1 promoter and the SceI target sequence.[6][7]
Technique Workflow
First the CORE cassette is amplified by PCR with primers containing regions of homology to the chromosomal site where it will be inserted. The CORE cassette is integrated via homologous recombination. Cells containing the CORE cassette can be selected for using the reporter gene and can be further confirmed using the counterselectable marker. Integration of the CORE cassette in the correct chromosomal location can be verified via PCR using primers that anneal upstream of the integration site, within the CORE and downstream of the integration size, which are designed to generate 500-1500 bp fragments.[4] CORE-containing yeast cells are transformed with oligonucleotides containing the desired mutation such that they lead to the loss of the CORE cassette during homologous recombination. Transformants are selected using the counterselectable marker and can be further screened using the reporter gene. Sequencing is used to ensure the correct mutation has been generated without additional mutations. Alternatively, if the mutation leads to the generation or loss of a restriction site, PCR followed by restriction digest can be used to confirm that the desired mutation has been integrated.[4]
Delitto perfetto
Delitto perfetto
References
[1] Erdeniz N., Mortensen UH., Rothstein R. (1997) Cloning-free PCR-based allele replacement methods. Genome Res. 7:1174-83. [2] Langle-Rouault F., Jacobs E. (1995) A method for performing precise alterations in the yeast genome using a recyclable selectable marker. Nucleic Acids Res. 23:3079-81. [3] Scherer S., Davis RW. (1979) Replacement of chromosome segments with altered DNA sequences constructed in vitro. Proc Natl Acad Sci. 76:4951-5. [4] Storici F., Resnick MA. (2006) The delitto perfetto approach to in vivo site-directed mutagenesis and chromosome rearrangements with synthetic oligonucleotides in yeast. Methods Enzymol. 409:329-45. [5] Inga A., Resnick MA. (2001) Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactive tumor p53 mutants. Oncogene. 14;20(27):3573-9 [6] Storici F., Durham CL., Gordenin DA., Resnick MA. (2003) Chromosomal site-specific double-strand breaks are efficiently targeted for repair by oligonucleotides in yeast. Proc Natl Acad Sci. 9;100(25):14994-9 [7] Plessis A., Perrin A., Haber JE., Dujon B. (1992) Site-specific recombination determined by I-SceI, a mitochondrial group I intron-encoded endonuclease expressed in the yeast nucleus. Genetics. 130(3):451-60 [8] Storici F., Resnick MA. (2003) Delitto perfetto targeted mutagenesis in yeast with oligonucleotides. Genet Eng. 25:189-207 [9] Storici F., Lewis LK., Resnick MA. (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol. 19(8):773-6
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