2021 Articles
Unbiased profiling of CRISPR RNA-guided transposition products by long-read sequencing
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.
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Also Published In
- Title
- Mobile DNA
- DOI
- https://doi.org/10.1186/s13100-021-00242-2
More About This Work
- Published Here
- September 22, 2023
Notes
CRISPR-Cas, RNA-guided transposase, Tn7, Transposition, SMRT-seq, CAST