The Sleeping Beauty transposase system is a tool for genetic engineering that introduces specific DNA sequences into the chromosomes of cells. It is a synthetic two-part system, consisting of a transposase enzyme and a DNA segment known as a transposon. The transposase is an enzyme that facilitates the movement of the transposon, often called a “jumping gene,” from one genomic location to another by cutting it from its original spot and inserting it elsewhere.
The Reawakening of a ‘Fossil’ Gene
The Sleeping Beauty system originates from non-functional genetic remnants found in the DNA of various species. These “fossil” genes are inactivated transposons that have accumulated mutations over millions of years, rendering them unable to produce the enzyme needed to move. In the 1990s, scientists hypothesized that they could reconstruct a working transposase from these dormant pieces.
Their research focused on the genomes of salmonid fish, which contain numerous inactive transposons from the Tc1/mariner family. By comparing these mutated sequences, researchers pieced together a consensus sequence for a functional transposase gene. They synthesized this reconstructed gene in the laboratory, bringing a long-extinct enzyme back to life.
This resurrected gene was named “Sleeping Beauty” because it was awakened from a prolonged evolutionary slumber. The creation of the first Sleeping Beauty transposase in 1997 provided a tool that could efficiently function in vertebrate cells and offered a new, non-viral method for genetic modification.
How the Sleeping Beauty System Works
The mechanism of the Sleeping Beauty system relies on two components: the transposon and the transposase enzyme. The transposon is a piece of DNA that contains the genetic material of interest, or “cargo,” flanked by specific recognition sequences called inverted repeats. These repeats act as handles for the transposase enzyme.
The process is a “cut-and-paste” operation. The Sleeping Beauty transposase enzyme identifies and binds to the inverted repeats on both ends of the transposon. Once attached, the enzyme cuts the transposon, excising it from its original location, which is often a carrier molecule called a plasmid.
With its genetic cargo, the transposase-transposon complex moves to a new location within the cell’s chromosomal DNA. The transposase searches for a specific, two-base-pair DNA sequence known as a TA dinucleotide site. Upon finding such a site, the enzyme cuts the cell’s DNA and pastes the transposon into the new position, resulting in the stable insertion of the cargo gene.
Applications in Gene Therapy and Research
The ability to permanently insert genetic material makes the Sleeping Beauty system a tool in research and medicine. It is used for treating various genetic disorders by delivering a functional copy of a faulty gene.
One application is in cancer treatment, specifically in the creation of CAR-T cells. In this form of immunotherapy, a patient’s own T-cells are genetically modified to recognize and attack cancer cells. The Sleeping Beauty system is used to insert a gene into the T-cells that produces a Chimeric Antigen Receptor (CAR) on their surface. This receptor binds to specific proteins on cancer cells, programming the T-cells to destroy them.
The system is also used to create animal models for studying human diseases. By introducing specific genes into the genome of a lab animal, such as a mouse, researchers can replicate the conditions of a genetic disorder. This allows them to study the disease’s progression and test potential treatments in a controlled setting.
Advancements and Safety Considerations
A primary concern with any technology that inserts DNA into a genome is the risk of “insertional mutagenesis.” This occurs if the transposon is pasted into a location that disrupts an important native gene, potentially leading to unintended consequences like the activation of a cancer-causing gene. The Sleeping Beauty system tends to insert into TA sites distributed somewhat randomly, so the possibility of landing in a problematic spot exists.
To improve the system’s performance, scientists have engineered enhanced versions of the transposase. Hyperactive variants have been developed, with one of the most notable being SB100X, a version up to 100 times more efficient at transposition than the original.
This increased efficiency is a safety enhancement. Because the hyperactive transposase works so well, smaller amounts of the enzyme and transposon are needed to achieve the desired genetic modification. Using lower doses reduces the overall number of insertion events, which in turn lowers the statistical probability of an insertion occurring in a sensitive region of the genome.