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Canonical prokaryotic type I CRISPR-Cas adaptive immune systems contain a multicomponent effector complex called Cascade, which degrades large stretches of DNA via Cas3 helicase-nuclease activity. Recently, a highly precise subtype I-F1 CRISPR-Cas system (HNH-Cascade) was found that lacks Cas3, the absence of which is compensated for by the insertion of an HNH endonuclease domain in the Cas8 Cascade component. Here, we describe the cryo-EM structure of Selenomonas sp. HNH-Cascade (SsCascade) in complex with target DNA and characterize its mechanism of action. The Cascade scaffold is complemented by the HNH domain, creating a ring-like structure in which the unwound target DNA is precisely cleaved. This structure visualizes a unique hybrid of two extensible biological systems-Cascade, an evolutionary platform for programmable DNA effectors, and an HNH nuclease, an adaptive domain with a spectrum of enzymatic activity., Competing Interests: Declaration of interests S.H., H.A.-T., and F.Z. are coinventors on a patent application (PCT/US2023/070150) related to this work filed by the Broad Institute and MIT. F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Aera Therapeutics, and Moonwalk Biosciences. F.Z. is a scientific advisor for Octant., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

To spread, transposons must integrate into target sites without disruption of essential genes while avoiding host defense systems. Tn7-like transposons employ multiple mechanisms for target-site selection, including protein-guided targeting and, in CRISPR-associated transposons (CASTs), RNA-guided targeting. Combining phylogenomic and structural analyses, we conducted a broad survey of target selectors, revealing diverse mechanisms used by Tn7 to recognize target sites, including previously uncharacterized target-selector proteins found in newly discovered transposable elements (TEs). We experimentally characterized a CAST I-D system and a Tn6022-like transposon that uses TnsF, which contains an inactivated tyrosine recombinase domain, to target the comM gene. Additionally, we identified a non-Tn7 transposon, Tsy, encoding a homolog of TnsF with an active tyrosine recombinase domain, which we show also inserts into comM. Our findings show that Tn7 transposons employ modular architecture and co-opt target selectors from various sources to optimize target selection and drive transposon spread., Competing Interests: Declaration of interests F.Z. is a scientific advisor and cofounder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, Proof Diagnostics, and Aera Therapeutics. F.Z. is a scientific advisor for Octant. G.F., M.K., and F.Z. are co-inventors on a provisional patent application filed by Broad relating to this work., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Type VI CRISPR-Cas13 effector enzymes catalyze RNA-guided RNA cleavage and have been harnessed for various technologies, such as RNA detection, targeting, and editing. Recent studies identified Cas13bt3 (also known as Cas13X.1) as a miniature Cas13 enzyme, which can be used for knockdown and editing of target transcripts in mammalian cells. However, the action mechanism of the compact Cas13bt3 remains unknown. Here, we report the structures of the Cas13bt3-guide RNA complex and the Cas13bt3-guide RNA-target RNA complex. The structures revealed how Cas13bt3 recognizes the guide RNA and its target RNA and provided insights into the activation mechanism of Cas13bt3, which is distinct from those of the other Cas13a/d enzymes. Furthermore, we rationally engineered enhanced Cas13bt3 variants and ultracompact RNA base editors. Overall, this study improves our mechanistic understanding of the CRISPR-Cas13 enzymes and paves the way for the development of efficient Cas13-mediated transcriptome modulation technologies., Competing Interests: Declaration of interests F.Z. is a co-founder of Editas Medicine, Beam Therapeutics, Pairwise Plants, Arbor Biotechnologies, and Sherlock Biosciences. O.N. is a co-founder, board member, and scientific advisor of Curreio. R.N., S.K., H.A.-T., F.Z., H.N., and O.N. have filed a patent application related to this work., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)