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

Abstract

CRISPR-associated transposons (CASTs) co-opt CRISPR-Cas 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 show that CASTs instead co-opt defense-associated CRISPR arrays for horizontal transmission. A bioinformatic analysis shows that all CAST sub-types co-occur with defense-associated CRISPR-Cas systems. Using an E. coli quantitative transposition assay, we show that CASTs use CRISPR RNAs (crRNAs) from these defense systems for horizontal gene transfer. A high-resolution structure of the type I-F CAST-Cascade in complex with a type III-B crRNA 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 a crRNA-independent unguided mechanism, even when the S15 co-factor is over-expressed. Over-expressing S15 and the trans-activating CRISPR RNA (tracrRNA) or a single guide RNA (sgRNA) reduces, but does not abrogate, off-target integration for type V CASTs. Exploiting new spacers in defense-associated CRISPR arrays explains how CASTs horizontally transfer to new hosts. More broadly, this work will guide further efforts to engineer the activity and specificity of CASTs for gene editing applications.

Keywords: Tn7; g ene editing; mobile genetic element; transposon.

Figure 1:. CASTs co-occur when CRISPR defense systems.

(A) A bioinformatics workflow for annotating CRISPR defense systems that co-occur with CASTs in the same genome. Blue: CRISPR-associated genes; brown: transposase genes. (B) CAST CRISPR arrays are shorter than defense-associated CRISPR arrays in the same genomes. (C) CASTs frequently 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.

Figure 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. Guided transposition into lacZ is scored as white, chloramphenicol-resistant clones. The donor strain is 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 (F) confirms 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. Error bars are the standard deviation across three biological replicates. Scrambling either the repeat or spacer suppressed transposition below our detection limit of < 10⁶ cfus.

Figure 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) Close-up view of the direct repeat (gray) and its interactions with Cas6 (salmon). (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.

Figure 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. Black: DR from the native CAST CRISPR array; gray: a sequence-scrambled DR that preserved the wild type stem loop structure; other colors correspond to the schematic in (A).

Figure 5:. Dual methods of horizontal transmission by Type V CASTs.

(A) The small ribosomal protein S15 reduces overall transposition. (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.

Figure 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).