Molecular mechanism of the type I-B2 CRISPR-associated transposon system

<p dir="ltr">CRISPR-associated transposons (CASTs) are naturally evolved Tn7-like transposons that have co-opted CRISPR-Cas systems for RNA-guided DNA transposition. Because CASTs can mediate targeted DNA insertion through programmable guide RNAs, they hold great promise as tools for precise, large-payload genome engineering. Despite their potential, the molecular mechanisms governing their transposition, including target DNA recognition, transposon protein recruitment, and donor DNA insertion, are not yet fully understood. A comprehensive mechanistic understanding is critical for advancing the development of CASTs in both biotechnological and therapeutic contexts.</p><p dir="ltr">In this study, we used cryo-electron microscopy (cryo-EM) and biochemical assays to investigate the type I-B2 CAST from <i>Peltigera membranacea cyanobiont</i> 210A (<i>Pmc</i>CAST), which features both RNA-guided and protein-guided pathways. In the protein-guided pathway, four proteins (TnsA, TnsB, TnsC, and TnsD) mediate transposition, with TnsD recognizing the target site, TnsC acting as a regulator, and TnsAB catalyzing the insertion reaction. In the RNA-guided pathway, the CRISPR ribonucleoprotein complex <i>Pmc</i>Cascade binds target DNA and utilizes the non-sequence-specific TnsD homolog TniQ to recruit TnsABC.</p><p dir="ltr">Biochemical reconstitution of the RNA-guided pathway using purified components led to the discovery of a previously unrecognized <i>Pmc</i>Cascade subunit that is critical for transposition activity. Cryo-EM analyses of key assemblies, including the <i>Pmc</i>Cascade–TniQ–TnsC–DNA recruitment complex, revealed a DNA-induced conformational change in <i>Pmc</i>Cascade that facilitates TniQ engagement in the RNA-guided pathway. To visualize the fully assembled transpososome, we reconstituted the complete TnsABCD complex in the TnsD-guided pathway, composed of TnsABCD proteins, transposon ends, and target DNA, and determined its structure by cryo-EM. The structure revealed interactions between TnsA and TnsB, and highlighted the role of TnsC in recruiting TnsAB and assembling the transpososome. Together, this thesis provides high-resolution insights into CAST-mediated gene insertion and establishes a structural framework for engineering next-generation programmable gene integration tools.</p>