Conformational analysis by Cryo-EM yields a high resolution singular model of RNA-guided transposition by CRISPR-associated transposons

CRISPR-associated transposons or CASTs are mobile genetic elements that use CRISPR-Cas systems to integrate large DNA cargoes at target loci in an RNA-guided manner.

CASTs are a relatively new and exciting addition to the gene-editing toolbox, and exploratory studies in human cells suggest their potential to treat genetic disease regardless of the underlying mutation(s) by replacing the entire disease-causing gene with a healthy copy.

While data from structural experiments has provided some information about how CASTs integrate foreign DNA, the resolution provided in previous studies has not enabled the detailed understanding of their mechanisms that is necessary to fully explore and exploit their gene-editing potential.

To help close that gap, a team at the Novo Nordisk Foundation Centre for Protein Research used high-resolution cryoelectron microscopy and targeted mutagenesis to yield detailed characterisations of CAST complexes and several assembly intermediates. Their findings, which are published in Molecular Cell today, contribute to our understanding of CAST transposition and will help researchers to adapt CASTs for human gene-editing applications.

The team, led by Professor Guillermo Montoya, reports high-resolution cryoelectron microscopy (cryo-EM) structures of the reconstituted post-transposition complex of the type V-K CAST, together with different assembly intermediates that enables recapitulation of the integration complex formation. In brief, they show that transposition activity can be modulated and even augmented by mutating certain amino acid residues within the CAST TnsB-binding sites and TnsC hairpin loop, and they identify features of the CAST that govern distance between the PAM and the attachment sites.

Speaking with CRISPR Medicine News about the impact of the study's findings on therapeutic applications, Guillermo Montoya said: »Although we have a long way to go, we are quite excited because our initial gene-editing experiments using the new CAST variants with enhanced activity are already showing promising results in human cells«.

Read the full study in Molecular Cell here.