Sequential structural rearrangements at the PAM-distal site of a type I-F3 CRISPR-Cas effector enabling RNA-guided DNA transposition

Abstract

Some prokaryotes carry CRISPR-associated transposons (CASTs), Tn7-like elements that incorporate genes encoding CRISPR-Cas effectors. CAST insertion is directed by CRISPR-Cas effectors through RNA-guided DNA binding and interactions with transposition-associated proteins. Although efficient sequence-specific DNA integration requires both precise target DNA recognition and coordinated interactions between effectors and transposition-associated proteins, the underlying mechanism remains elusive. Here, we determined three cryo-EM structures of target DNA-bound type I-F3 TniQ-Cascade from Vibrio parahaemolyticus, revealing how Cas8/5 recognizes the protospacer adjacent motif (PAM) and identifying a key residue responsible for the cytidine preference at position -2 of the PAM. We revealed mismatch tolerance at the PAM-proximal site. Structural analyses showed that correct base pairing at the PAM-distal site correlates with conformational changes in the Cas8/5 helical bundle and TniQ, bending the DNA to guide its downstream region toward the transposition machinery. Together, these dynamic rearrangements at the PAM-distal region provide insights into the licensing mechanism of type I-F3 CAST transposition and highlight its potential for genome engineering applications.

Graphical Abstract

Figure 1.

Overall structure of type I-F3 TniQ-Cascade from V. parahaemolyticus. (A) Schematic diagram of the CAST in V. parahaemolyticus RIMD 2210633 (top) and the hflK gene on V. alginolyticus K08M03 plasmid pL300 (bottom). Nucleotide numberings for the yciA and hflK genes, as well as the protospacers (PSP) targeted by the first and the third crRNAs, are indicated. (B, C) Overall structures of target DNA-bound VpTniQ-Cascade in form 1 and form 2, respectively. (Left) Cryo-EM map of the complex. (Middle) Cartoon representation of the complex. The numbering of α-helices in the Cas8/5 HB is indicated. (Right) Stick representations of the target DNA and crRNA within the complex. (D) Schematic diagram of the R-loop structure between the crRNA derived from the first spacer of the CRISPR array in panel (A) and its perfectly matched target DNA. The crRNA, TS, and NTS are colored yellow, light green, and dark green, respectively. The PAM sequence is pink. Disordered nucleotides in the form 2 structure are colored gray. Nucleotide numberings of each strand are indicated.

Figure 2.

PAM recognition mechanism by Cas8/5 of VpTniQ-Cascade. (A) Structural comparison of the PAM recognition mechanism between the type I-F3 VpTniQ-Cascade (left) and the type I-F1 PaCascade (right; PDB ID: 6NE0). Cas8/5 of the VpTniQ-Cascade and Cas8 of the PaCascade are shown as cartoon representations. The TS and NTS are depicted as light green and dark green stick models, respectively. Dashed lines indicate hydrogen bonds. (B) In vivo transposition assay of the wild-type and Cas8/5 D126A mutant-containing VpTniQ-Cascade for 16 different PAM sequences. Transposition efficiencies are plotted. Data are presented as mean ± SD from three independent experiments. The mean transposition efficiency is indicated. nd, not detected.

Figure 3.

Effects of mismatched base pairs on transposition. (A) In vivo reconstitution of transposition into the native protospacer of the hflK gene. The schematic diagram depicts transposition into a target plasmid containing the hflK–hflC gene operon, resulting in transposed pInsert plasmids in both forward and reverse orientations (left). The positions of primer pairs and the sizes of PCR amplicons are indicated above the corresponding regions. The amplicons were analyzed by agarose gel electrophoresis (right). The gel image is representative of three independent experiments. (B) In vivo transposition efficiencies with crRNA and mismatched target DNA. Transposition efficiencies, evaluated by qPCR, are plotted. Data are presented as mean ± SD from three independent experiments. The mean transposition efficiency is indicated above each bar. nd, not detected. (C) Structural comparison of the seed region between the perfectly matched protospacer (top; form 1) and the hflK protospacer (bottom). The crRNA and the TS are depicted as stick representations. Cryo-EM maps for the first five base pairs of the heteroduplex in each structure are shown.(D) Structural comparison of the seed regions between the VpTniQ-Cascade containing mismatched base pairs (left) and PaCascade (right). Protein subunits are shown as surface models (top) and cartoon models (bottom). The Thr457 residue of Cas8/5 in VpTniQ-Cascade and its corresponding residue, Arg85 of Cas8 in PaCascade, are depicted as stick representations.

Figure 4.

Conformational changes of the Cas8/5 HB and TniQ in coordinating dsDNA accommodation at the PAM-distal region. (A) Domain architectures of Cas8/5 and TniQ (top). The secondary structures of the HB domain in Cas8/5 and the HTH2 domain in TniQ are highlighted (bottom). Amino acid residue numbering is indicated. (B–E) Overall structures of target DNA-bound TniQ-Cascade. Protein subunits and nucleic acids are shown as surface representations, with the six Cas7 subunits and TniQ.2 rendered transparent. Panels (B–E) correspond to the PR state, FR1 state, and FR2 state of VpTniQ-Cascade (this study) and the full R-loop state of AsTniQ-Cascade (PDB ID: 7U5D), respectively. (F–I) Close-up views of the structures at the PAM-distal site, indicated by dotted lines in panels (B–E), respectively. Panels (F–I) correspond to the PR state, FR1 state, and FR2 state of VpTniQ-Cascade, and the full R-loop state of AsTniQ-Cascade, respectively. The α-helices of the Cas8/5 HB and TniQ.1 HTH2 are labeled. The model is shown together with the cryo-EM focused map in panels F–H. (J) Detailed view of the interactions between the 8/5-loop and the Q-loop in the PR state, as indicated by dotted lines in panel (F). The atomic model is shown together with the cryo-EM focused map. Hydrophobic residues at the interface are shown in stick models. (K) In vivo transposition assay of wild-type VpTniQ-Cascade and its variants containing quadruple mutations in the 8/5-loop or Q-loop. Transposition efficiencies are shown as bar plots. Data are presented as mean ± SD from three independent experiments. The mean transposition efficiency is indicated above each bar. (L) Structural comparisons of the interface involving the Cas8/5 HB, the Q-loop, and the heteroduplex at the PAM-distal site between the PR and FR1 states (left) and between the PR and FR2 states (right). For these comparisons, the TniQ-Cascade structures were superimposed using Cas7.1 as the reference. Orange octagrams indicate steric clashes between the Q-loop in the PR state and the TS in the FR1 state (left), as well as between the Q-loop in the PR state and the TS in the FR2 state (right).

Figure 5.

Genome engineering in methionine auxotroph E. coli strain B834(DE3). (A) Schematic diagram of the in vivo methionine synthesis gene complementation experiment. The CRISPR array containing a single spacer targeting the sequence of the native promoter of the metE gene on pTniQ-Cascade and the corresponding target sequence on the genomic DNA of E. coli strain B834(DE3) are shown in light yellow. (B) Growth of each transformant on minimal medium in the presence or absence of methionine. The images of transformants are representative of three independent experiments. (C) Detection of TniQ-Cascade-dependent transposition. The DNA regions amplified by colony PCR and their corresponding product sizes are shown (top). The amplicons were analyzed by agarose gel electrophoresis (bottom). Tn and PC denote the fragment amplified from the transposed DNA into the genome and the positive control DNA fragment for PCR, respectively. The gel images are representative of three independent experiments.

Figure 6.

Model of transposition licensing via conformational changes at the PAM-distal site. In the PR state, accurate base pairing at positions 25–29 is monitored, which facilitates the following DNA bending through interactions with the Cas8/5 HB and TniQ.1 (top left). If mismatches are present in the PAM-distal region (mismatched positions 25–29), then proper DNA bending would likely not occur, and as a result, the transposition reaction does not take place (bottom left). The Cas8/5 HB and the two TniQ molecules undergo rigid-body rearrangements to enlarge the space at the TS binding site, enabling the formation of a full R-loop (the FR1 state; top middle). Once the TS reaches TniQ.1, the hydrophobic interactions between the Cas8/5 HB and TniQ.1 are rearranged, resulting in the rotation of the Cas8/5 HB and bringing both ends of TniQ-Cascade closer together (the FR2 state; top right). This structural rearrangement is proposed to stabilize the downstream dsDNA for subsequent reactions. Through these licensing processes, interactions with transposition-associated proteins would be facilitated (bottom right).