Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition

• Published in: Molecular cell 2023-06, Vol.83 (11), p.1827-1838.e6
• Main Authors: Park, Jung-Un, Petassi, Michael T., Hsieh, Shan-Chi, Mehrotra, Eshan, Schuler, Gabriel, Budhathoki, Jagat, Truong, Vinh H., Thyme, Summer B., Ke, Ailong, Kellogg, Elizabeth H., Peters, Joseph E.
• Format: Article
• Language: English
• Subjects: Cascade-TniQ; CAST; CRISPR-Associated Proteins - genetics; CRISPR-associated transposon; CRISPR-Cas; CRISPR-Cas Systems; cryo-EM; DNA - genetics; guide RNA-directed; I-F3 CAST; PAM recognition; R-loop; RNA; RNA-guided integration; Tn7; transposition
• ISSN: 1097-2765; 1097-4164
• DOI: 10.1016/j.molcel.2023.05.005

CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition. [Display omitted] •Structures of I-F3 CAST effector module reveal roles of transposon protein TniQ•TniQ participates in complete R-loop formation by engaging with target DNA•TniQ regulates target-site choice through interactions with crRNA•Structural basis of PAM ambiguity in I-F3 CAST compared with canonical CRISPR-Cas Park et al. investigate the mechanisms of the DNA-recognition module from I-F3 CRISPR-Cas-associated transposons, Cascade-TniQ. They demonstrate that TniQ functions in target engagement and regulation of target-site choice with Cascade components. Comparing PAM recognition of I-F3 Cascade with canonical CRISPR-Cas reveals the structural basis of PAM ambiguity.