New insights into CRISPR-associated transposons published in Nature Communications

A study led by Branco Weiss Fellow Irma Querques, who is affiliated with the Max Perutz Labs in Vienna, has been published in Nature Communications. The research provides new insights into the molecular mechanisms of type I-F CRISPR-associated transposons (CASTs), which are a promising tool for genome editing.

Transposons are often known as “jumping” genes because they are DNA sequences that can move from one genomic location to another. Bacterial CRISPR-associated transposons combine this ability with CRISPR-based RNA guidance, which enables a targeted insertion of DNA without creating double-strand breaks. This makes CASTs promising candidates for programmable gene insertion.

However, despite their potential, much remains unknown about the functional mechanism required for precise DNA transposition. In the new study, Querques and her colleagues worked with type I-F CASTs, which show high activity in human cells, and focused specifically on the Pseudoalteromonas type I-F CAST transposase TnsAB.

By combining cryo-electron microscopy with biochemical assays, the researchers resolved different stages of the transposition reaction at an atomic level and described a series of molecular checkpoints that control how and when the transposase acts. They found that the transposase initially recognizes the ends of the transposon with surprisingly high sequence specificity before correctly assembling into an active complex capable of precisely excising the transposon DNA sequence.

They also demonstrated that the left and right transposon ends play a dual role: they define the cleavage sites and promote assembly of the transposase complex. But assembly alone is not sufficient for cleavage: the complex must be activated upon target engagement, providing a final checkpoint before transposon excision. These tightly coordinated steps ensure that the transposition process occurs with high precision and help explain how the “cut” and “paste” stages of the reaction are linked.

Together, the findings provide a mechanistic framework for understanding how type I-F CASTs achieve precise DNA transposition and advance our fundamental understanding of this molecular system. This work could lay the groundwork for the further development of more precise and predictable genome-editing tools.

Read the article in Nature Communications: Transposon end recognition and excision mechanisms of type I-F CRISPR-associated transposases

Read the press release by Max Perutz Labs: Cut-and-paste, but not cut, then paste