Abstract
Bacterial transposons propagate through either non-replicative (cut-and-paste) or replicative (copy-and-paste) pathways, depending on how the mobile element is excised from its donor source. In the well-characterized E. coli transposon Tn7, a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each transposon end during the excision step. Whether a similar pathway is involved for RNA-guided transposons, in which CRISPR-Cas systems confer DNA target specificity, has not been determined. Here, we apply long-read, population-based whole-genome sequencing (WGS) to unambiguously resolve transposition products for two evolutionarily distinct transposon classes that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results reveal that RNA-guided transposon systems lacking functional TnsA primarily generate cointegrate products containing genomically inserted vector backbones. Finally, we report natural and engineered transposon variants encoding a TnsAB fusion protein, revealing a novel strategy for achieving RNA-guided transposition with fewer molecular components.
Competing Interest Statement
P.L.H.V. and S.H.S. are inventors on patents and patent applications related to CRISPR-Cas systems and uses thereof. S.H.S. is a co-founder and scientific advisor to Dahlia Biosciences, and an equity holder in Dahlia Biosciences and Caribou Biosciences.