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

• Published in: Mobile DNA Vol. 12; no. 1; pp. 1 - 13
• Main Authors: Vo, Phuc Leo H, Acree, Christopher, Smith, Melissa L, Sternberg, Samuel H
• Format: Journal Article
• Language: English
• Published: London BioMed Central Ltd 08.06.2021; BioMed Central; BMC
• Subjects: CAST; CRISPR; CRISPR-Cas; E coli; Enzymes; Escherichia coli; Genomes; Mutation; Plasmids; Polypeptides; Population; Proteins; RNA; RNA-guided transposase; Short Report; SMRT-seq; Tn7; Transposition; Transposons
• ISSN: 1759-8753; 1759-8753
• DOI: 10.1186/s13100-021-00242-2

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 types that employ either Cascade or Cas12k for RNA-guided DNA integration. Our results show that RNA-guided transposon systems lacking functional TnsA primarily undergo copy-and-paste transposition, generating cointegrate products that comprise duplicated transposon copies and genomic insertion of the vector backbone. 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.