Structures of the holo CRISPR RNA-guided transposon integration complex

Abstract

CRISPR-associated transposons (CAST) are programmable mobile genetic elements that insert large DNA cargos using an RNA-guided mechanism1–3. CAST elements contain multiple conserved proteins: a CRISPR effector (Cas12k or Cascade), a AAA+ regulator (TnsC), a transposase (TnsA–TnsB) and a target-site-associated factor (TniQ). These components are thought to cooperatively integrate DNA via formation of a multisubunit transposition integration complex (transpososome). Here we reconstituted the approximately 1 MDa type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined its structure using single-particle cryo-electon microscopy. The architecture of this transpososome reveals modular association between the components. Cas12k forms a complex with ribosomal subunit S15 and TniQ, stabilizing formation of a full R-loop. TnsC has dedicated interaction interfaces with TniQ and TnsB. Of note, we observe TnsC–TnsB interactions at the C-terminal face of TnsC, which contribute to the stimulation of ATPase activity. Although the TnsC oligomeric assembly deviates slightly from the helical configuration found in isolation, the TnsC-bound target DNA conformation differs markedly in the transpososome. As a consequence, TnsC makes new protein–DNA interactions throughout the transpososome that are important for transposition activity. Finally, we identify two distinct transpososome populations that differ in their DNA contacts near TniQ. This suggests that associations with the CRISPR effector can be flexible. This ShCAST transpososome structure enhances our understanding of CAST transposition systems and suggests ways to improve CAST transposition for precision genome-editing applications.

Distinct horizontal transfer mechanisms for type I and type V CRISPR-associated transposons

“…These results are consistent with our earlier bioinformatic observation of multi-site insertion in other type V systems (Figure S6) (Rybarski et al, 2021). We conclude that type V CASTs do not require any CRISPR array to mobilize via unguided transposition (Querques et al, 2021; Xiao et al, 2021; Wang et al, 2022; Tenjo-Castaño et al, 2022; Park et al, 2022). Taken together, these results highlight that type V CASTs are much more permissive than either type I-F or I-B systems.…”

“…The crRNA contained a native direct repeat from the type III-B system and a 32 bp spacer. The density for Cascade and the crRNA was refined with a prior model (PDB: 6PIG) (Halpin-Healy et al, 2020; Jia et al, 2020; Li et al, 2020; Park et al, 2022). The overall structure was quite similar to the prior model ( C A − RMSD = 0.83Å), indicating the native DR from the type III-B can assemble a functional Cascade (Figure 3).…”

“…As before, we removed the CAST’s homing spacer and targeted a single guide RNA (sgRNA) or the native tracr/crRNA to lacZ . We also tested whether the E. coli or S. hofmannii small ribosomal subunit protein S15 aides transposition (Park et al, 2022; Schmitz et al, 2022). Transposition efficiency was scored by measuring the colony forming units on chloramphenicol-resistant plates.…”

“…Transposition was dependent on cas12k , as its ablation dropped transposition below our detection limit ( < 10 − 7 cfus). Recent structural studies identified the small ribosomal protein S15 as a core CAST subunit that increases the on-target transposition rate (Park et al, 2022; Schmitz et al, 2022). Surprisingly, expressing either E. coli or S. hofmannii S15 (EcS15 or ShS15) reduced transposition ∼ 50–250 fold, respectively.…”

IS21 family transposase cleaved donor complex traps two right-handed superhelical crossings

“…Several classic mobile elements, including Tn7 and bacteriophage Mu, also depend on ATP-dependent factors to select the appropriate insertion sites 70 , 72 – 74 . Recently, the structures of a recruitment complex and a holo-transposome from a Tn7-like element have been reported 75 , 76 . Although these studies help to define how the AAA + factor selects the insertion site, the structure of the TnsB tetramer remains largely unchanged and, therefore, how these ‘molecular matchmakers’ precisely regulate the transposase activity to promote DNA transposition remains unclear.…”