The completed sequencing of entire bacterial genomes and recent advances in genomics has allowed the identification of many genes. While several genes have been characterized, the function of the majority remains unknown. Bridging the gap between sequence identification and gene function will aid in the understanding of many physiological processes and genetic regulation mechanisms.
One important tool that enables the unraveling of gene function is targeted gene disruption. This knockout technology enables mutations to be rapidly generated in a specific gene or genes and is critical for defining biological function of a given gene and for characterizing regulatory networks.
Gene knockouts for generating mutant prokaryotes are frequently produced by transposon mutagenesis. Transposons are small DNA segments that have the ability to move from one position in the genome to another.
Also referred to as "jumping genes," these elements can insert into the bacterial genome and cause mutations that disrupt genes in a nontargeted manner. This process is catalyzed by the enzyme transponase, which in most cases is expressed from a gene carried on the transposon.
An Alternative, Targeted Method
Mobile introns are self-splicing RNAs and retrotransposable elements found in bacteria and lower eukaryote organelles. These can insert into specific DNA target sites with high frequency. Group II introns are released in a lariat form that can efficiently integrate into intronless alleles of the same gene.
This process is called homing and depends on the expression of intron-encoded proteins possessing a variety of functions including maturase (intron splicing), DNA binding, endonuclease, and reverse transcriptase activities.
Homing is initiated when the intron-encoded protein forms a complex in cis with the intron RNA that recognize a specific DNA sequence in the genome. Since group II intron homing proceeds through a reverse-transcribed copy of the RNA intron, the process has been termed retrohoming.
Recent advances in group II intron research have enabled the efficient and specific re-targeting of introns. Based on a group II intron from Lactococcus lactis, the recently developed TargeTron Gene Knockout System, commercialized by Sigma-Aldrich (St. Louis), provides a novel functional genomics tool for targeted and permanent gene disruption.
Protocols have been optimized that allow insertional knockouts in E. coli within only three days. Unlike conventional transposon mutagenesis methods, group II introns are site-specific, not random.
How the Technology Works
Group II introns insert themselves via the activity of an RNA-protein complex (RNP) expressed from a single plasmid. The RNA portion of the RNP is easily mutated via PCR to re-target insertion into a user-specified chromosomal gene (Figure 1).
As an analogy, group II introns can be likened to programmable restriction enzymes, but with the added activity of inserting RNA into a cleaved DNA sequence. TargetTron uses a simple, streamlined protocol that identifies target sites in the gene of interest using computer algorithms.
For example, a typical 1 kb gene can be expected to contain approximately eight group II intron insertion sites allowing mutagenesis throughout the length of an open reading frame. The algorithm is located on the TargeTron design website (www.sigma-aldrich.com/ targetronaccess) and requires only the sequence of the target gene to be entered to generate gene specific primers.
The program provides the user with suggested oligo sequences for mutating the group II intron sequence (Figure 2). The oligos are designed for specific points of insertion and are ranked in order of expected efficiency. After selection of one or more insertion points, the user must order the required oligos. A test site allows genes to be prescreened for potential insertion sites before investing in the new system.
One or more of the primer set options may be used to mutate (re-target) the RNA intron component by PCR. After PCR has been performed, the resulting PCR fragment is ligated into a linearized vector that contains the remaining intron components. The ligation reaction is transformed into the host and the re-targeted intron is expressed.
The resulting mutated RNP complex scans the host genomic DNA for the specific insertion site. The RNA intron inserts into the gene of interest followed by reverse transcription and repair to create a permanent, double-strand DNA disrupted mutant.
Knockouts are then selected using a kanamycin marker that is activated upon chromosomal insertion. Using gene-specific primers, kanamycin-resistant colonies are PCR screened to confirm insertion (Figure 3).
Advantages of the TargeTron System
Numerous characteristics of the group II retrohoming mechanism make it attractive for genetic manipulation. First, retrohoming is highly efficient, enabling creation of knockouts without the need for a selection marker. To complement the current kanamycin-based TargeTron system (TA0100), Sigma plans to release a series of TargeTron pACD4 vectors that do not insert antibiotic markers. These will allow researchers to expedite the creation of multiple knockouts.
Second, the introns have a minimal dependence on host factors, making them applicable to a broad range of bacteria. To date, the TargeTron system has been validated in a wide range of bacteria including: E. coli, Staphylococcus aureus, Clostridium, Shigella flexneri, and Salmonella typhimurium. The system may be modified for use in additional organisms.
In addition to site-specific knockouts, random chromosomal insertion libraries can be created. This approach allows essential and nonessential genes to be identified by PCR and sequencing. Once a desired random insertion is identified by PCR, the gene-specific intron sequence can be cloned and used to re-create the site-specific knockout in hosts with alternative genetic backgrounds.
This eliminates the need for maintaining large clonal libraries to isolate random insertion events and also eliminates the need for phage-based transfer of mutations between strains.
In addition to knocking genes out by insertional mutagenesis, the TargeTron system can be modified to deliver heterologous DNA. This feature allows the use of retrotransposition-activated selectable markers (RAM).
The currently available TargeTron system uses the same RAM approach, but with a kanamycin marker. This feature is not limited to the use of selectable markers and was recently used to examine the use of the TargeTron system to introduce therapeutic sequences site specifically into mutant genes.
The ability to target specific genomic regions and deliver heterologous DNA is a powerful combination allowing for the study of various chromosomal regions using promoters, reporters, and other genetic elements.
In summary, compared to existing knockout methodologies for prokaryotes, the TargetTron system allows efficient, targeted disruption of the gene of interest in a wide range of bacteria. It has significant applications in genetic engineering and metabolic engineering in systems biology and functional genomics.