Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsB's preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.

Soai's asymmetric autocatalysis represents a highly remarkable example for spontaneous symmetry breaking and enantioselective amplification in the enantioselective alkylation of pyrimidine‐5‐carbaldehydes to the corresponding chiral pyrimidine alcohols. Recently, zinc hemiacetalate complexes, formed from pyrimidine‐5‐carbaldehydes and the chiral product alcohol, were identified by in situ high‐resolution mass spectrometric measurements as highly active transient asymmetric catalysts in this autocatalytic transformation. To study the formation of such hemiacetals and their stereodynamic properties, we focused on the synthesis of coumarin homolog biaryl systems with carbaldehyde and alcohol substituents. Such systems are able to form hemiacetals by intramolecular cyclization. An interesting feature of the substituted biaryl backbone is that tropos and atropos systems can be obtained, enabling or disabling the intramolecular cyclization to hemiacetals. Biaryl structures with various functional groups were synthesized, and the equilibrium and stereodynamics between the closed and open structures were investigated by dynamic enantioselective HPLC (DHPLC). The enantiomerization barriers ΔGǂ and activation parameters ΔHǂ and ΔSǂ were determined from temperature dependent kinetic measurements.