Sleeping Beauty transposon system

The Sleeping Beauty transposon system is a made-up piece of DNA that helps add new pieces of DNA into the chromosomes of animals with backbones. It is used to give animals new traits or find new genes and learn what they do. It belongs to a group called Tc1/mariner, and the main tool called transposase was made from old, inactive fish DNA.

How it works

Figure 1. How SB transposon works: Top line: The transposon is shown with red double arrows (IR/DRs) inside another piece of DNA, like a plasmid (blue lines). The transposon contains an expression cassette with a promoter (blue oval) that controls how the gene or other DNA part called "genetic cargo" is used. Middle lines: The Sleeping Beauty (SB) transposase sticks to the IR/DRs, then cuts the transposon out of the plasmid. The black slashes show where the cuts happen in the plasmid. Bottom lines: A different piece of DNA (green) with a TA sequence is where the transposon can be placed. When it moves, the TA sequence is copied at the new spot.

The Sleeping Beauty system has two parts: the Sleeping Beauty transposase and a transposon. It was created in 1997 to insert specific pieces of DNA into the genomes of animals with backbones. DNA transposons move by cutting from one spot in the DNA and pasting into another spot (see Fig. 1). This moving process is very exact — a piece of DNA is cut out and moved to another place in the same or different DNA.

Like other Tc1/mariner transposases, the Sleeping Beauty transposase puts the transposon into a TA base pair (a tiny part of DNA) in the target DNA. The new spot for the transposon can be in the same piece of DNA or in a different one. In the DNA of mammals like humans, there are about 200 million TA spots. When the transposon goes into a TA spot, it copies the TA part. This copying is a special sign that helps scientists figure out how it works. A new study shows that sometimes it can go into non-TA spots, but this is rare.

The transposase can be inside the transposon (like in Fig. 2), or it can come from somewhere else. If it comes from outside, the transposon cannot move again by itself. Transposons that cannot move by themselves (like in Fig. 1) are better for genetic research because they stay in place after being added. In human and other mammal genomes, all transposons are the kind that cannot move by themselves. Even though they have transposase genes, these genes do not work, so they cannot move the transposon.

Construction

Figure 2. Parts of SB transposase: The SB transposase has 360 amino acids and three main parts. The first part (at the start) is the DNA-recognition domain, which grabs the DRs in the IR/DRs of the transposon. It has two paired box shapes, called PAI and RED, that can hold onto DNA. The second part is the NLS, which helps the transposase move into the nucleus where it works. The third part is the DDE domain, which cuts and pastes DNA. The DDE domain has special DDE (or sometimes DDD) amino acids, found in many enzymes like transposases and recombinases. There is also a part filled with glycine (G) amino acids.

The transposase gene was named "Sleeping Beauty (SB)" because it was brought back to life after being inactive for a long time. The SB transposon system is synthetic, meaning it was made by humans. The SB transposase was built using old (fossil) pieces of transposase from a group called Tc1/mariner found in the DNA of salmon fish. In humans, about 3% of our DNA comes from Tc1/mariner transposons, but they stopped working more than 10 million years ago because of changes called mutations. Scientists guessed that there was an original Tc1-like transposon that was the ancestor of the ones in fish. Even though many transposon sequences were found in fish, they could not move because of mutations. By looking at how the sequences changed in different fish, scientists guessed what the original transposon might have been like (Fig. 2).

To build the transposase, scientists combined parts of two transposons from Atlantic salmon and one from rainbow trout. They fixed small problems in the enzyme so it could work (Fig. 3). The first working version, called SB10, was created using a “majority-rule” method, choosing the most common amino acid from 12 genes in eight fish. The first steps (1 to 3 in Fig. 3) filled in missing parts and fixed stop signals that would block the making of a 360-amino acid protein. The next step (4) repaired a signal that moves the transposase from the cytoplasm (where it is made) to the nucleus (where it works). Steps 5 to 8 fixed the start of the enzyme, which helps it recognize repeats (DRs) in the DNA. The last steps repaired the part that cuts and pastes DNA, using key amino acids found in similar enzymes. SB10 had everything needed to work.

Figure 3. How SB transposase was made: Step 1: Diagram of old Tc1/mariner transposons from modern salmonid fish. Mutations are marked: x for missense, S for stop signals, F for frameshifts, and G for gaps where amino acids are missing. Step 3: Fixing gaps, stop signals, and frameshift mutations. Step 4: Rebuilding the NLS sequence (orange underline). Steps 5–8: Rebuilding the start (N-terminal) DNA-binding part (orange underline). Steps 9–10: Rebuilding the DDE domain (orange underline) with its DDE amino acids (green boxes).

Since SB10, scientists made better versions by testing more changes and using more transposon sequences. The transposase binds to DNA using two parts called PAI and RED. PAI is most important for recognizing repeats, while RED overlaps with the signal for moving into the nucleus, though its role is unclear. The newest version, SB100X, works 100 times better than SB10. In 2009, SB100X was named "Molecule of the Year" by a scientific group because of its future potential in gene editing.

The transposon used by SB transposase was called T because it was found in the fish Tanichthys albonubes. T has a special sequence with inverted repeats (IRs) that contain smaller direct repeats (DRs) (see Figs. 1 and 2). T’s IR/DR sequence was the closest match to the original Tc1 transposons. The IRs are 231 base pairs, while the DRs are 29 and 31 base pairs. This length difference is important for fast transposition. The T transposon has been improved by small changes to better match other related transposons.

Applications

Figure 4. Uses of Sleeping Beauty transposon system

For the past 10 years, SB transposons have been used to add genes to the DNA of animals with backbones and for gene therapy. The gene added can be part of a special setup called an expression cassette, which controls how much of the gene is made and in which body part it works. Another way to use SB transposons is to learn what genes do, especially genes that cause cancer, by adding pieces of DNA that stop nearby genes from working. This is called insertional mutagenesis or transposon mutagenesis. When a gene is turned off by a transposon or another method, it is called a “knockout.” Scientists have made knockout mice and rats using the SB system (Fig. 4).

For adding genes or turning them off, SB transposons work like viruses but are not viruses. Viruses are very good at getting inside cells and copying themselves, but cells have strong defenses to block them. For some gene therapy uses, avoiding viruses is better for safety and rules. Non-viral systems like SB transposons skip many of the defenses that block viruses.

Plasmids are small, round pieces of DNA (shown in Fig. 1) often used to deliver genes without viruses. But plasmids have two big problems when used to put genes into DNA. First, the added genes do not last long because they do not stay part of the cell’s DNA. Second, it is hard to get plasmids into cells. The Sleeping Beauty system solves the first problem. SB transposons add DNA directly into the genome (Fig. 1), so the gene stays active for a long time, even in future generations. Also, SB transposons avoid making extra copies that can stop the gene from working. Adding genes with plasmids is still less effective than using viruses, but strong promoters can help make enough gene product for an entire animal, even if only a few cells receive the transposon.

The most exciting future use of SB transposons may be for treating human diseases with gene therapy. If the system is cheap, it can be used in rich and poor countries alike. Since the SB system only uses DNA, it costs much less to make and deliver than viruses. The first tests of SB transposons in gene therapy will use modified T cells to help patients with severe cancer.