Delitto perfetto

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Delitto perfetto (Italian:  [deˈlitto perˈfɛtto] ) is a genetic technique for in vivo site-directed mutagenesis in yeast. This name is the Italian term for "perfect murder", and it refers to the ability of the technique to create desired genetic changes without leaving any foreign DNA in the genome.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

Genome entirety of an organisms hereditary information; genome of organism (encoded by the genomic DNA) is the (biological) information of heredity which is passed from one generation of organism to the next; is transcribed to produce various RNAs

In the fields of molecular biology and genetics, a genome is the genetic material of an organism. It consists of DNA. The genome includes both the genes and the noncoding DNA, as well as mitochondrial DNA and chloroplast DNA. The study of the genome is called genomics.

Contents

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).

The National Institute of Environmental Health Sciences (NIEHS) conducts research into the effects of the environment on human disease, as one of the 27 institutes and centers of the National Institutes of Health (NIH).

Oligonucleotides are short DNA or RNA molecules, oligomers, that have a wide range of applications in genetic testing, research, and forensics. Commonly made in the laboratory by solid-phase chemical synthesis, these small bits of nucleic acids can be manufactured as single-stranded molecules with any user-specified sequence, and so are vital for artificial gene synthesis, polymerase chain reaction (PCR), DNA sequencing, library construction and as molecular probes. In nature, oligonucleotides are usually found as small RNA molecules that function in the regulation of gene expression, or are degradation intermediates derived from the breakdown of larger nucleic acid molecules.

Chromosomal crossover

Chromosomal crossover is the exchange of genetic material between 2 homologous chromosomes non-sister chromatids that results in recombinant chromosomes during sexual reproduction. It is one of the final phases of genetic recombination, which occurs in the pachytene stage of prophase I of meiosis during a process called synapsis. Synapsis begins before the synaptonemal complex develops and is not completed until near the end of prophase I. Crossover usually occurs when matching regions on matching chromosomes break and then reconnect to the other chromosome.

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.

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 transfection or other procedure meant to introduce foreign DNA into a cell. Selectable markers are often antibiotic resistance genes. Bacteria that have been subjected to a procedure to introduce foreign DNA are grown on a medium containing an antibiotic, and those bacterial colonies that can grow have successfully taken up and expressed the introduced genetic material. Normally the genes encoding resistance to antibiotics such as ampicillin, chloroamphenicol, tetracycline or kanamycin, etc., are considered useful selectable markers for E. coli.

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]

DNA sequencing biological research program

DNA sequencing is the process of determining the nucleic acid sequence – the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine, guanine, cytosine, and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery.

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. In addition, human genes can be studied and similarly genetically manipulated in yeast by using yeast artificial chromosomes (YACs).

<i>Escherichia coli</i> species of Gram-negative, rod-shaped bacterium

Escherichia coli, also known as E. coli, is a Gram-negative, facultative anaerobic, rod-shaped, coliform bacterium of the genus Escherichia that is commonly found in the lower intestine of warm-blooded organisms (endotherms). Most E. coli strains are harmless, but some serotypes can cause serious food poisoning in their hosts, and are occasionally responsible for product recalls due to food contamination. The harmless strains are part of the normal microbiota of the gut, and can benefit their hosts by producing vitamin K2, and preventing colonization of the intestine with pathogenic bacteria, having a symbiotic relationship. E. coli is expelled into the environment within fecal matter. The bacterium grows massively in fresh fecal matter under aerobic conditions for 3 days, but its numbers decline slowly afterwards.

Genetic engineering Direct manipulation of an organisms genome using biotechnology

Genetic engineering, also called genetic modification or genetic manipulation, is the direct manipulation of an organism's genes using biotechnology. It is a set of technologies used to change the genetic makeup of cells, including the transfer of genes within and across species boundaries to produce improved or novel organisms. New DNA is obtained by either isolating and copying the genetic material of interest using recombinant DNA methods or by artificially synthesising the DNA. A construct is usually created and used to insert this DNA into the host organism. The first recombinant DNA molecule was made by Paul Berg in 1972 by combining DNA from the monkey virus SV40 with the lambda virus. As well as inserting genes, the process can be used to remove, or "knock out", genes. The new DNA can be inserted randomly, or targeted to a specific part of the genome.

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.

<i>Saccharomyces cerevisiae</i> species of fungus

Saccharomyces cerevisiae is a species of yeast. It has been instrumental to winemaking, baking, and brewing since ancient times. It is believed to have been originally isolated from the skin of grapes. It is one of the most intensively studied eukaryotic model organisms in molecular and cell biology, much like Escherichia coli as the model bacterium. It is the microorganism behind the most common type of fermentation. S. cerevisiae cells are round to ovoid, 5–10 μm in diameter. It reproduces by a division process known as budding.

RAD52 protein-coding gene in the species Homo sapiens

RAD52 homolog , also known as RAD52, is a protein which in humans is encoded by the RAD52 gene.

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

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.

Figure 1. CORE Cassettes for Delitto Perfetto Cassettes.png
Figure 1. CORE Cassettes for Delitto Perfetto

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

Counterselectable markers

Additional features

Figure 2. Overview ofDelitto Perfetto for Gene Deletion Delitto Perfetto method for gene deletions.png
Figure 2. Overview ofDelitto Perfetto for Gene Deletion

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]

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 repair machinery. [8] This increases the frequency of targeted homologous recombination by 4,000 fold compared to when no DSB is generated. [6]

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. [4] [9] 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. [9]

For gene deletions

Oligonucleotides containing the sequence upstream immediately followed by the sequence downstream of the region to be knocked out are designed. For gene deletions, pairs of fully overlapping 80–100 bp oligonucleotides lead to 5–10 fold increase in transformation efficiency than single oligonucleotides. [4]

For point mutations

For mutations 20 to 40 bp from the CORE cassette, 80–100 bp fully overlapping oligonucleotides are recommended. For mutations more than 40 bp from the CORE cassette, partly overlapping oligonucleotides must be used. To increase their transformation efficiency, it is recommended that partly overlapping oligonucleotides be extended in vitro. [4] [6] [9]

For essential genes

To generate mutants of essential genes, the CORE cassette can be inserted downstream of the gene of interest, however this limits the regions of the gene available for mutation. Alternatively, diploid cells can be used. However, using a diploid decreases the efficiency of oligonucleotide targeting due to the presence of two suitable chromosomal locations for the oligonucleotides to recombine. To address this drawback, the DSB-mediated delitto perfetto method can be used. This increases the frequency of targeted homologous recombination by 700 fold compared to when no DSB is generated. Moreover, it is 2–5 fold more efficient than other available methods. [6] [9]

Background mutation rates

It is reported that when no double stranded breaks are generated, the number of cells that lose the CORE cassette in the absence of a targeting oligonucleotide is less than one transformant per 107 viable cells. In contrast, this number increases up to 100 transformants per 107 viable cells when a double stranded break is generated. In a diploid, the increased background mutation rates occur due to homologous recombination with the homologous chromosome decreasing targeted transformation events to only 4% of the total transformants. [6]

Related Research Articles

A gene knockout is a genetic technique in which one of an organism's genes is made inoperative. However, KO can also refer to the gene that is knocked out or the organism that carries the gene knockout. Knockout organisms or simply knockouts are used to study gene function, usually by investigating the effect of gene loss. Researchers draw inferences from the difference between the knockout organism and normal individuals.

Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles. It is also a product and services market, with an estimated value of $168 billion by 2017.

Cloning vector

A cloning vector is a small piece of DNA, taken from a virus, a plasmid, or the cell of a higher organism, that can be stably maintained in an organism, and into which a foreign DNA fragment can be inserted for cloning purposes. The vector therefore contains features that allow for the convenient insertion or removal of a DNA fragment to or from vector, for example by treating the vector and the foreign DNA with a restriction enzyme that cuts the DNA. DNA fragments thus generated contain either blunt ends or overhangs known as sticky ends, and vector DNA and foreign DNA with compatible ends can then be joined together by molecular ligation. After a DNA fragment has been cloned into a cloning vector, it may be further subcloned into another vector designed for more specific use.

This is a list of topics in molecular biology. See also index of biochemistry articles.

Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.

Mitotic recombination is a type of genetic recombination that may occur in somatic cells during their preparation for mitosis in both sexual and asexual organisms. In asexual organisms, the study of mitotic recombination is one way to understand genetic linkage because it is the only source of recombination within an individual. Additionally, mitotic recombination can result in the expression of recessive genes in an otherwise heterozygous individual. This expression has important implications for the study of tumorigenesis and lethal recessive genes. Mitotic homologous recombination occurs mainly between sister chromatids subsequent to replication. Inter-sister homologous recombination is ordinarily genetically silent. During mitosis the incidence of recombination between non-sister homologous chromatids is only about 1% of that between sister chromatids.

Recombineering is a genetic and molecular biology technique based on homologous recombination systems, as opposed to the older/more common method of using restriction enzymes and ligases to combine DNA sequences in a specified order. Recombineering is widely used for bacterial genetics, in the generation of target vectors for making a conditional mouse knockout, and for modifying DNA of any source often contained on a bacterial artificial chromosome (BAC), among other applications.

Sister chromatid exchange

Sister chromatid exchange (SCE) is the exchange of genetic material between two identical sister chromatids.

FLP-FRT recombination

In genetics, Flp-FRT recombination is a site-directed recombination technology, increasingly used to manipulate an organism's DNA under controlled conditions in vivo. It is analogous to Cre-lox recombination but involves the recombination of sequences between short flippase recognition target (FRT) sites by the recombinase flippase(Flp)derived from the 2 µm plasmid of baker's yeast Saccharomyces cerevisiae.

Balancer chromosomes are special, modified chromosomes used for genetically screening a population of organisms to select for heterozygotes. Balancer chromosomes can be used as a genetic tool to prevent crossing over between homologous chromosomes during meiosis. Balancers are most often used in Drosophila melanogaster genetics to allow populations of flies carrying heterozygous mutations to be maintained without constantly screening for the mutations but can also be used in mice. Balancer chromosomes have three important properties: they suppress recombination with their homologs, carry dominant markers, and negatively affect reproductive fitness when carried homozygously.

Tetrad (meiosis) the four spores of a yeast, other Ascomycota or Chlamydomonas produced after meiosis

The tetrad is the four spores produced after meiosis of a yeast or other Ascomycota, Chlamydomonas or other alga, or a plant. After parent haploids mate, they produce diploids. Under appropriate environmental conditions, diploids sporulate and undergo meiosis. The meiotic products, spores, remain packaged in the parental cell body to produce the tetrad.

Transposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

Knockout rat

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.

A knockout mouse or knock-out mouse is a genetically modified mouse in which researchers have inactivated, or "knocked out", an existing gene by replacing it or disrupting it with an artificial piece of DNA. They are important animal models for studying the role of genes which have been sequenced but whose functions have not been determined. By causing a specific gene to be inactive in the mouse, and observing any differences from normal behaviour or physiology, researchers can infer its probable function.

Mutagenesis in the laboratory is an important technique whereby DNA mutations are deliberately engineered to produce mutant genes, proteins, strains of bacteria, or other genetically modified organisms. Various constituents of a gene, such as its control elements and its gene product, may be mutated so that the functioning of a gene or protein can be examined in detail. The mutation may also produce mutant proteins with interesting properties, or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of particular cell function to be investigated.

Cassette mutagenesis is a type of site-directed mutagenesis that uses a short, double-stranded oligonucleotide sequence to replace a fragment of target DNA. It uses complimentary restriction enzyme digest ends on the target DNA and gene cassette to achieve specificity. It is different from methods that use single oligonucleotide in that a single gene cassette can contain multiple mutations. Unlike many site directed mutagenesis methods, cassette mutagenesis also does not involve primer extension by DNA polymerase.

Synthetic genome is a synthetically-built genome whose formation involves either genetic modification on pre-existing life forms or artificial gene synthesis to create new DNA or entire lifeforms.. The field that studies synthetic genomes is called Synthetic Genomics.

SCAR-less genome editing Scarless Cas9 Assisted Recombineering (no-SCAR) is an editing method that is able to manipulate the Escherichia coli genome. The system relies on recombineering whereby DNA sequences are combined and manipulated through homologous recombination. No-SCAR is able to manipulate the E. coli genome without the use of the chromosomal markers detailed in previous recombineering methods. Instead, in this method, the λ-Red recombination system facilitates donor DNA integration while Cas9 cleaves double-stranded DNA to counter-select against wild-type cells. Although λ-Red and Cas9 genome editing are widely used technologies, the no-SCAR method is novel in combining the two functions; this technique is able to establish point mutations, gene deletions, and short sequence insertions in several genomic loci with increased efficiency and time sensitivity.

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. 1 2 3 4 5 6 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. 1 2 3 4 5 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. 1 2 3 4 Storici F., Lewis LK., Resnick MA. (2001) In vivo site-directed mutagenesis using oligonucleotides. Nat Biotechnol. 19(8):773-6
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