Delitto perfetto
Encyclopedia
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
.
(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 oligonucleotide
s 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).
s 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 .
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).
, 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.
There are a variety of CORE cassettes to choose from, which contain a variety of reporter genes, counterselectable makers and additional features.
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.
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.
machinery. This increases the frequency of targeted homologous recombination by 4,000 fold compared to when no DSB is generated.
Longer oligonucleotides lead to increased transformation efficiency. Fully complementary oligonucleotides pairs lead to 5-10 fold increase in efficiency compared to single oligonucleotides and are recommended for all applications. However, they provide a small window of mutagenesis of only 20-40 bases from the CORE cassette. To increase the window of mutagenesis, oligonucleotide pairs with a 20 bp overlap can be used, and these allow up to 100 bp upstream and downstream of the CORE integration site to be targeted. However, they transform approximately 6 times less efficiently. To increase the efficiency, partly overlapping oligonucleotides can be extended in vitro.
Site-directed mutagenesis
Site-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
in yeast. This name is an Italian term for perfect deletion and is also an idiom
Idiom
Idiom is an expression, word, or phrase that has a figurative meaning that is comprehended in regard to a common use of that expression that is separate from the literal meaning or definition of the words of which it is made...
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
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
.
Background
This technique was developed by a group at the National Institute of Environmental Health SciencesNational Institute of Environmental Health Sciences
The National Institute of Environmental Health Sciences is a part of the National Institutes of Health , which is in turn a part of the Department of Health and Human Services ....
(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 oligonucleotide
Oligonucleotide
An oligonucleotide is a short nucleic acid polymer, typically with fifty or fewer bases. Although they can be formed by bond cleavage of longer segments, they are now more commonly synthesized, in a sequence-specific manner, from individual nucleoside phosphoramidites...
s in combination with the cellular process of homologous recombination
Chromosomal crossover
Chromosomal crossover is an exchange of genetic material between homologous chromosomes. It is one of the final phases of genetic recombination, which occurs during prophase I of meiosis in a process called synapsis. Synapsis begins before the synaptonemal complex develops, and is not completed...
. 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 markerSelectable marker
A selectable marker is a gene introduced into a cell, especially a bacterium or to cells in culture, that confers a trait suitable for artificial selection. They are a type of reporter gene used in laboratory microbiology, molecular biology, and genetic engineering to indicate the success of a...
s 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
DNA sequencing
DNA sequencing includes several methods and technologies that are used for determining the order of the nucleotide bases—adenine, guanine, cytosine, and thymine—in a molecule of DNA....
to confirm mutagenesis, which is often a complicated and inefficient process .
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
Escherichia coli
Escherichia coli is a Gram-negative, rod-shaped bacterium that is commonly found in the lower intestine of warm-blooded organisms . Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in humans, and are occasionally responsible for product recalls...
. analysis In addition, human genes can be studied and similarly genetically manipulated
Genetic engineering
Genetic engineering, also called genetic modification, is the direct human manipulation of an organism's genome using modern DNA technology. It involves the introduction of foreign DNA or synthetic genes into the organism of interest...
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 cerevisiaeSaccharomyces cerevisiae
Saccharomyces cerevisiae is a species of yeast. It is perhaps the most useful yeast, having been instrumental to baking and brewing since ancient times. It is believed that it was originally isolated from the skin of grapes...
, the RAD52
RAD52
RAD52 homolog , also known as RAD52, is a protein which in humans is encoded by the RAD52 gene.- Function :The protein encoded by this gene shares similarity with Saccharomyces cerevisiae Rad52, a protein important for DNA double-strand break repair and homologous recombination...
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.
Technical drawbacks
- Cost of oligonucleotides
- Mutagenesis is limited to region of genome surrounding the inserted cassette
- Limited number of reporter geneReporter geneIn molecular biology, a reporter gene is a gene that researchers attach to a regulatory sequence of another gene of interest in cell culture, animals or plants. Certain genes are chosen as reporters because the characteristics they confer on organisms expressing them are easily identified and...
s (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.
Reporter genes
- kanMX4 - allows for growth in media containing GeneticinG418G418 is an aminoglycoside antibiotic similar in structure to gentamicin B1. It is produced by Micromonospora rhodorangea. G418 blocks polypeptide synthesis by inhibiting the elongation step in both prokaryotic and eukaryotic cells...
- hyg - allows for growth in media containing Hygromycin BHygromycin BHygromycin B is an antibiotic produced by the bacterium Streptomyces hygroscopicus. It is an aminoglycoside that kills bacteria, fungi and higher eukaryotic cells by inhibiting protein synthesis.- History :...
.
Counterselectable markers
- KlURA3URA3URA3 is gene on Chromosome V in Saccharomyces . Its systematic name is YEL021W.URA3 is a gene that encodes orotidine 5-phosphate decarboxylase , which is an enzyme that catalyzes one reaction involved in the synthesis of pyrimidine ribonucleotides in yeast RNA.Loss of ODCase activity leads to a...
- prevents growth in media containing 5-flouroorotic acid. - GAL1/10-p53 – prevents growth in media containing galactoseGalactoseGalactose , sometimes abbreviated Gal, is a type of sugar that is less sweet than glucose. It is a C-4 epimer of glucose....
. It encodes a toxic mutant of p53P53p53 , is a tumor suppressor protein that in humans is encoded by the TP53 gene. p53 is crucial in multicellular organisms, where it regulates the cell cycle and, thus, functions as a tumor suppressor that is involved in preventing cancer...
under a GAL1 promoter .
Additional features
- GAL1-I-SceI – Increases the efficiency of targeting to the CORE cassette-containing chromosome in diploid cells. It contains the restriction endonucleaseRestriction enzymeA Restriction Enzyme is an enzyme that cuts double-stranded DNA at specific recognition nucleotide sequences known as restriction sites. Such enzymes, found in bacteria and archaea, are thought to have evolved to provide a defense mechanism against invading viruses...
SceI under the GAL1 promoter and the SceI target sequence .
Technique Workflow
First the CORE cassette is amplified by PCRPolymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
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.
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
Mutation
In molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
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.
Double-strand break (DSB) mediated delitto perfetto
To increase oligonucleotide targeting to the CORE cassette, CORE cassettes containing the GAL1-I-SceI feature can be used. This feature allows for the expression of SceI, an endonuclease that recognizes a highly unique 18 nucleotide sequence unlikely to occur anywhere else in the S. cerevisiae genome. The SceI endonuclease is able to generate a DSB at the SceI site leading to the recruitment of the DNA repairDNA repair
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. In human cells, both normal metabolic activities and environmental factors such as UV light and radiation can cause DNA damage, resulting in as many as 1...
machinery. This increases the frequency of targeted homologous recombination by 4,000 fold compared to when no DSB is generated.
General Considerations for Oligonucleotide Design
80-100 bp oligonucleotides can be generated as single molecules, or pairs of oligonucleotides that are completely overlapping or partially overlapping. The type of oligonucleotide recommended depends on the type of mutation and the distance from the CORE cassette integration site a mutation is desired.Longer oligonucleotides lead to increased transformation efficiency. Fully complementary oligonucleotides pairs lead to 5-10 fold increase in efficiency compared to single oligonucleotides and are recommended for all applications. However, they provide a small window of mutagenesis of only 20-40 bases from the CORE cassette. To increase the window of mutagenesis, oligonucleotide pairs with a 20 bp overlap can be used, and these allow up to 100 bp upstream and downstream of the CORE integration site to be targeted. However, they transform approximately 6 times less efficiently. To increase the efficiency, partly overlapping oligonucleotides can be extended in vitro.