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

Abstract

CASTs use both CRISPR-associated proteins and Tn7-family transposons for RNA-guided vertical and horizontal transmission. CASTs encode minimal CRISPR arrays but can't acquire new spacers. Here, we report that CASTs can co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that CASTs co-occur with defense-associated CRISPR systems, with the highest prevalence for type I-B and type V CAST sub-types. Using an E. coli quantitative transposition assay and in vitro reconstitution, we show that CASTs can use CRISPR RNAs from these defense systems. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B CRISPR RNA reveals that Cas6 recognizes direct repeats via sequence-independent π - π interactions. In addition to using heterologous CRISPR arrays, type V CASTs can also transpose via an unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA or a single guide RNA reduces, but does not abrogate, off-target integration for type V CASTs. Our findings suggest that some CASTs may exploit defense-associated CRISPR arrays and that this fact must be considered when porting CASTs to heterologous bacterial hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Fig. 1. CASTs co-occur with defense-associated CRISPR systems.

A A bioinformatics workflow for annotating CRISPR defense systems that co-occur with CASTs in the same genome. Blue: CRISPR-associated genes (cas); brown: transposase genes (tns). B CAST CRISPR arrays are shorter than defense-associated CRISPR arrays in the same genomes. C CASTs co-exist with one or more additional CRISPR arrays. D Defense-associated CRISPR-Cas sub-types that co-exist with CASTs in the NCBI microbial genome database.

Fig. 2. Type I-F CASTs co-opt defense-associated CRISPR arrays.

A The predicted structures of direct repeats (DRs) from a type I-F CAST and co-occurring defense CRISPR-Cas systems. Blue: 4-5 nt loop; green: 5-7 bp stem; yellow: 5'- and 3'-handles. B Schematic of a quantitative conjugation-based mate-out transposition assay. A plasmid harboring the CAST, along with the cargo antibiotic resistance (green), and a minimal CRISPR array is conjugated into the recipient strain and plated on the indicated Luria-Bertani (LB) agar plates. Guided transposition into lacZ is scored as white, chloramphenicol-resistant (Cmr) clones. The donor strain MFDpir+ is a conjugative, DAP auxotrophic, Mu-free donor strain of E. coli designed to support the replication of R6k suicide vectors which will be removed via counter-selection with diaminopimelic acid (DAP). C Direct repeats from the defense associated CRISPR arrays support transposition, but a scrambled direct repeat does not. D Colony-resolved long-read sequencing (E) and Sanger sequencing confirm cut-and-paste transposition into lacZ (triangle in E). Target site duplication (TSD) is also visible in this data. F Quantification of transposition from the native CAST array and co-occurring defense systems, in colony forming units (CFU). Error bars are the standard deviation across three biological replicates. Scrambling either the repeat or spacer suppressed transposition below our detection limit of  < 10⁶ CFU.

Fig. 3. Cas6 recognizes the crRNA via sequence-independent interactions with the DR.

A Structural overview of a type I-F TniQ-Cascade purified with a type III-B crRNA. B Magnified view of Cas6 (salmon) interacting with the direct repeat (gray). F138 interacts with the sole flipped nucleotide, C54, at the tip of the direct repeat through a stacking interaction. An arginine-rich helix stabilizes the entire stem-loop structure via a network of interactions along the sugar-phosphate backbone. C Schematic of the hydrophobic and electrostatic interactions between key Cas6 residues and the direct repeat from the type III-B crRNA. D Multiple sequence alignment across all CAST I-F cas6 genes reveals conserved residues in the arginine-rich helix. E Transposition requires Cas6 residues R121, R125, R129, and F138 to coordinate the direct repeat. (F)Quantification of transposition from CAST I-F system with mutated Cas6. Error bars are the standard deviation across three biological replicates.

Fig. 4. The crRNA stem-loop length regulates transposition activity.

A We tested changes in the direct repeat sequence, stem (green), loop (blue), and handle lengths (5'-orange, 3'-yellow). B The effect of each feature on the integration efficiency. Data are shown as mean ± S.D., n = 3 biologically independent experiments. Black: DR from the native CAST CRISPR array; gray: a sequence-scrambled DR that preserved the wild-type stem loop structure; and other colors correspond to the schematic in (A).

Fig. 5. Dual methods of horizontal transmission by Type V CASTs.

A The small ribosomal protein S15 reduces overall transposition. Data are shown as mean ± S.D., n = 3 biologically independent experiments. B Over-expression of E. coli S15 stimulates on-target transposition, even with a defense-associated type I-D crRNA and the sgRNA. However, significant off-target transposition remains even when S15 is over-expressed. C Long-read sequencing with the type I-D crRNA confirms that most, but not all, insertions are in lacZ when ShS15 is over-expressed. Triangle: target site. The target site duplication (TSD, gray) and left and right inverted repeats (blue) are also visible at the insertion site. (bottom). D The cargo is inserted  ~35–42 bp away from the target site (purple in B). E Schematic (left) and quantification (right) of simple insertion and co-integration products via long-read sequencing. Over-expressing S15 and using heterologous crRNA does not significantly alter this ratio. cmR: chloramphenicol resistance gene; tracr: trans-activating crRNA; CR: CRISPR array; R, L: left and right inverted repeats.

Fig. 6. Dual strategies for horizontal transmission by type I and type V CASTs.

All CASTs co-opt defense associated CRISPR arrays (red) for horizontal transmission. These arrays are updated by defense-associated Cas1-Cas2 integrases. Type V CASTs can also integrate non-specifically via crRNA-independent transposition. Both systems use a homing spacer for vertical transmission (purple).