Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing

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 well-characterized E. coli transposon Tn 7 , a heteromeric TnsA-TnsB transposase directs cut-and-paste transposition by cleaving both strands at each end during excision step. Whether similar pathway 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 products two evolutionarily distinct types that employ Cascade Cas12k integration. Our results show lacking functional TnsA primarily undergo copy-and-paste transposition, generating cointegrate comprise duplicated copies and genomic insertion of vector backbone. Finally, report natural engineered variants encoding TnsAB fusion protein, revealing novel strategy achieving with fewer molecular components.