2025 Theses Doctoral

Discovery of programmable RNA-guided mechanisms for transposition and gene regulation

Hoffmann, Florian T.

CRISPR-Cas systems are widespread in bacteria and archaea, providing potent mechanisms foradaptive immunity. Their reprogrammable, RNA-guided nucleic acid targeting and cleavage capabilities have been harnessed to create high-precision, next-generation genome editing tools. Although CRISPR-Cas systems are thought to have evolved from transposon-encoded nucleases, the extent to which these homologous proteins can perform RNA-guided functions, and how these systems affect transposition, remain largely unexplored. Moreover, numerous transposon-derived nucleases and Cas homologs are found alongside novel genes unrelated to bacterial immunity.

These associations, often involving nuclease-inactivated Cas-like proteins, suggest co-option and neofunctionalization of RNA-guided DNA targeting capabilities. Through bioinformatic analyses, multiple such systems were identified, unveiling a diverse repertoire of repurposed CRISPR-Cas- like pathways. Next-generation sequencing, combined with biochemical and structural approaches, elucidated the mechanisms by which CRISPR-associated transposons (CASTs) achieve efficient DNA integration and revealed how transposon-encoded Cas homologs facilitate transposon spread.

Furthermore, novel RNA-guided regulators were discovered that either repress or activate gene expression in a fully programmable manner. Strikingly, these RNA-guided activation systems operate independently of canonical promoter motif constraints, expanding our understanding of bacterial transcription. This work underscores the functional versatility of RNA-guided systems, highlighting their roles in pathways beyond antiviral immunity. These discoveries provide a roadmap for developing enhanced gene-editing technologies and novel tools enabling programmable gene expression.