Sleeping Beauty

    DNA transposons are mobile genetic elements that can recognize and copy or move their DNA to another genomic location using an encoded enzyme called transposase. Most of them rely on a “cut-and-paste” transposition mechanism driven by transposase, which is essentially a nuclease with a catalytic domain and at least one DNA-binding domain for the recognition of Terminal Inverted Repeats (TIR) at the transposon ends.

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    Inactive transposon copies were discovered in fish genomes in 1997 and were revived through rational reconstruction from inactive copies and named Sleeping Beauty (SB). Rounds of optimization led to the SB100x version, which is now widely used in genetic engineering to insert genetic material into eukaryotic genomes. It integrates with virtually no preference for genomic location, strictly requiring only a TA dinucleotide as a target site. This is a huge advantage compared to other transposases and viral integrases, which often prefer Transcription Start Sites or active genes.

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    Chimeric Antigen Receptor T cell (CAR-T) generation is a prominent example of using SB as a non-viral and safer method to genetically modify patient-derived T cells. Such CAR-T cells can recognize and kill specific cancer cells. CAR-T cell personalized immunotherapy is a very promising treatment for various types of blood cancer. Currently, there are a few clinical trials running worldwide to evaluate safety and efficiency.

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    Since its discovery in 1997, there have been numerous attempts to determine the atomic structure of SB transposition assemblies (transpososome). Despite its broad use, the SB DNA complex has proven to be a difficult target for structure determination, limiting our understanding of its mechanism and rational advancements in genetic applications.

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    When I joined the project, I dug through the literature and numerous lab books to assess what had been done and find the missing aspects preventing the crystallization of the SB transpososome. It looked very challenging to attempt structure determination again, and I was even advised against it. But I had a feeling, like in iconic X-Files series slogan - "The Truth Is Out There", and I could find it. Through rational optimization of DNA substrates and the creation of a custom crystallization screen, I succeeded and saw the first crystals. Nobody could believe it, and there were many doubts, but X-ray diffraction “points”, as the French say, “mettre les points sur les i”. The first structure of the SB transpososome was unveiled!

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    After that, new SB transpososomes continued to surprise me. During one experiment, I encountered a massive precipitation with a particular DNA substrate, but I couldn’t discard it and left it over the Christmas break in the fridge. To my astonishment, after the break, the tube had a huge crystal over 1 mm in size. The delicate task of retrieving this crystal from the tube's bottom in the cold room was nerve-wracking. To my surprise, it was still diffracting, and I could get the structure at low resolution and later on establish controlled crystallization.