y Dear Editor, CRISPR-Cas systems provide adaptive immunity in prokaryotes through RNA-guided cleavage of mobile genetic elements and have been harnessed as powerful genome editing tools. A programmable guide RNA and the effector Cas protein are delivered to mammalian cells to create double strand breaks on the target sequence in the genome, triggering endogenous DNA repair pathways that enable genetic changes. However, recent studies revealed transposon-associated CRISPR-Cas systems that are inactive in target cleavage but direct transposases for RNAguided DNA transposition. [1][2][3][4] Those findings open a new paradigm for precise DNA insertion independent of DNA repair pathways. 1,2 RNA-guided DNA integration in type I-F CRISPR-Cas system from Vibrio cholerae requires the DNA-targeting Cascade complex (also called the Csy complex) that is composed of Cas6, Cas7, a natural fusion of Cas8 and Cas5 (Cas8/5), and a 60-nt crRNA, along with the transposition machinery including TnsA, TnsB, TnsC and TniQ (a homolog of TnsD) (Fig. 1a). TniQ binds to the Vibrio cholerae Cascade complex (VcCascade) and recruits the core TnsABC machinery for transposition, thus playing an essential role in transposition. To understand the molecular basis if the RNA-guided DNA integration process, we determined the structure of VcCascade bound to TniQ at 3.1 Å resolution by cryo-EM (Fig. 1b, c; Supplementary information, Figs. S1-3, and Table S1). The resultant maps are of sufficient quality for accurate atomic model building for VcCascade and TniQ, except for the helical bundle of Cas8/5 which is built as a poly-alanine model (Fig. 1b).The architecture of VcCascade adopts a "G" shape (Fig. 1b), similar to that of Cascade from Pseudomonas aeruginosa (PaCascade) [5][6][7][8] (Supplementary Information Fig. S4a-d). Cas6, Cas7 and Cas8/5 with a stoichiometry of 1:6:1 are integrated by the crRNA, where the spacer sequence is flanked by a 3′ hairpin structure and a 5′ handle (Supplementary information, Fig. S4e). Six copies of Cas7 (Cas7.1-Cas7.6) assemble into a right-handed helical backbone with each Cas7 corresponding to a twist of ~46°a nd a rise of ~13 Å (Fig. 1b, c; Supplementary information, Fig. S4f). The structure of Cas7 is similar to that of Cas7 from PaCascade, with a root-mean-square deviation (RMSD) of 1.6 Å (Supplementary information, Fig. S4g). The Cas7 backbone is capped by Cas6 associated with the crRNA 3′ hairpin, forming the head of VcCascade (Fig. 1b, c; Supplementary information, Fig. S4h). Although the overall folds of Cas6 from VcCascade and PaCascade are similar, substantial differences were observed (RMSD = 5.6 Å; Supplementary information, Fig. S4i), indicative of distinct functional roles (to be discussed below). In the tail of VcCascade, Cas7 backbone is terminated by binding of Cas8/5 to the crRNA 5′ handle (Fig. 1b, c; Supplementary Information Fig. S4j). Cas8/5 consists of an N-terminal domain (Cas8/5 NTD ), a middle helical bundle (Cas8/5 HB ) and a C-terminal domain (Cas8/5 CTD ), which are homologous to Ca...

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Cas12f, also known as Cas14, is an exceptionally small type V-F CRISPR–Cas nuclease that is roughly half the size of comparable nucleases of this type. To reveal the mechanisms underlying substrate recognition and cleavage, we determined the cryo-EM structures of the Cas12f-sgRNA-target DNA and Cas12f-sgRNA complexes at 3.1 and 3.9 Å, respectively. An asymmetric Cas12f dimer is bound to one sgRNA for recognition and cleavage of dsDNA substrate with a T-rich PAM sequence. Despite its dimerization, Cas12f adopts a conserved activation mechanism among the type V nucleases which requires coordinated conformational changes induced by the formation of the crRNA-target DNA heteroduplex, including the close-to-open transition in the lid motif of the RuvC domain. Only one RuvC domain in the Cas12f dimer is activated by substrate recognition, and the substrate bound to the activated RuvC domain is captured in the structure. Structure-assisted truncated sgRNA, which is less than half the length of the original sgRNA, is still active for target DNA cleavage. Our results expand our understanding of the diverse type V CRISPR–Cas nucleases and facilitate potential genome editing applications using the miniature Cas12f.