GSE183114
Selective recruitment of the AAA+ ATPase TnsC increases the fidelity of Type I-F CRISPR RNA-guided transposition
Bacterial transposons are pervasive mobile genetic elements that exploit distinct DNA binding proteins for their horizontal spread. For example, E. coli Tn7 homes to a specific attachment site using a TniQ family protein, whereas diverse Tn7-like transposons employ Type I or Type V CRISPR-Cas systems to insert downstream of target sites specified by a guide RNA. Despite this targeting pathway diversity, transposition invariably requires TnsB, a DDE superfamily transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between the transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained elusive. Here we apply chromatin immunoprecipitation sequencing (ChIP-seq) to monitor in vivo formation of the Vibrio cholerae RNA-guided transpososome, allowing us to unambiguously resolve distinct protein recruitment events prior to integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous, leading to binding at hundreds of genomic off-target sites, but only a subset of those sites are licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint that controls transposition fidelity. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we analysed V. cholerae TnsC by cryo-EM and found that TnsC forms ATP-dependent heptameric rings, which are likely to play a critical architectural role in positioning DNA substrates for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive binding of distinct factors to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts. Overall design: E. coli genome ChIP-seq profiles for 5 proteins from the CRISPR type-IF-associated transposon. 31 samples were sequenced in total. The raw read files "Input_Cas8_crRNA_ChIP-seq_paired_raw" and "Input_Cas8_crRNA-287_ChIP-seq_paired_raw" are control samples which were used as reference samples for MACS3 peak calling. Replicate samples are indicated as "rep1" or "rep2". Samples that were re-sequenced in order to obtain more sequencing depth are named "[...]_more-depth".