Modularity and diversity of target selectors in Tn7 transposons

Mechanism of target site selection by type V-K CRISPR-associated transposases

“…Although there is currently no direct evidence to support this hypothesis, recent experiments with a native type V-K CAST system in cyanobacteria indicate that expression of Cas12k and TnsB are regulated by a CvkR transcriptional repressor ( 30 ), a feature that could be important in modulating the choice between targeted and untargeted transposition pathways. Considering the highly conserved operonic nature of TnsB, TnsC, and TniQ in Tn 7 -like elements, Tn 5053 , and type V-K CASTs, tight regulation in the stoichiometry of these proteins could be important for accessing an RNA-independent untargeted pathway ( 13 ). Future studies will be necessary to investigate this hypothesis further by deep sequencing bacteria with native type V-K elements to ensure that these rare events are not missed.…”

“…Although insertion specificity is often dictated by a single recombinase enzyme ( 2 , 3 ), some transposons encode heteromeric transposase complexes that distribute DNA target and integration activities across multiple distinct molecular components ( 4 , 5 ). Tn 7 -like transposons are unique in this regard, in that they have evolved to exploit diverse molecular pathways for target site selection, including site-specific DNA-binding proteins ( 6 ), replication fork–specific DNA-binding proteins ( 7 – 9 ), CRISPR RNA–guided DNA binding complexes ( 10 – 12 ), and additional DNA targeting pathways that have yet to be characterized ( 13 ). CRISPR-associated transposases (CASTs), in particular, represent both a fascinating example of CRISPR-Cas exaptation and an opportune starting point for the development of next-generation tools for programmable, large-scale DNA insertion ( 14 , 15 ).…”

Targeted Transcriptional Activation using a CRISPR-Associated Transposon System

“…This includes the commonly used Scytonema hofmanni type V-K CAST, 39 a large variety of type I-F CAST systems, 34,40,41 as well as some type I-B and I-D CAST systems. [42][43][44] These and other systems can provide alternatives with different PAM preferences, transposition mechanisms, and insertion behaviors, expanding the possibilities for applying CAST systems as transcriptional perturbators.…”

Targeted Transcriptional Activation Using a CRISPR-Associated Transposon System

“…Additionally, the bioinformatic analysis of genomes has uncovered a diversity of CAST systems that could be adapted for our approach. This includes the commonly used Scytonema hofmanni-type V–K CAST, a large variety of type I–F CAST systems, ,, as well as some type I–B and I–D CAST systems. − These and other systems can provide alternatives with different PAM preferences, transposition mechanisms, and insertion behaviors, expanding the possibilities for applying CAST systems as transcriptional perturbators.…”