Biology subjects

Kellogg, E. H.

Publications and source records attributed to Kellogg, E. H..

8 recordsLinked to original sources

bioRxiv2026

Transposon end recognition and pairing by I-F3 CRISPR-associated transposase

To develop gene therapy tools based on CRISPR-associated transposons (CASTs), it is essential to define how transposon ends are recognized and paired during transposition. Tn7-like transposons typically contain asymmetric left- and right-end sequences that flank and define DNA cargo. However, how the transposase recognizes these different sequences and assembles them into a paired end complex for cut-and-paste transposition remains unknown. Here we present the cryo-EM structure of type I-F3 (VchCAST) CAST transposase TnsB in complex with transposon DNA ends and host factor IHF, along with biochemistry, molecular dynamics, and in vivo analyses. Our structure reveals the stoichiometry and architecture of the assembly, as well as the DNA distortions required to accommodate transposon end asymmetry. Molecular dynamics suggests that these distortions are required for the coordinated assembly of the complex. Physical pairing of asymmetric left and right ends results in a novel protein-protein interface that is required for transposition efficiency. Our findings explain how transposases regulate the pairing of transposon end sequences for high-fidelity recognition, reveal a model of transposon end synapsis, and suggest future avenues to engineer DNA cargo for genome-editing applications.

biochemistry↗

bioRxiv2026

Structure of the Type I-F3 CAST holo integration complex reveals licensing mechanisms during RNA-guided DNA integration

Precise genomic integration of large DNA cargo remains a central challenge in genome engineering. In this context, CRISPR-associated transposases (CASTs) offer a promising solution by coupling RNA-guided DNA recognition to transposon-mediated integration. Type I-F3 CASTs exhibit exceptional targeting fidelity, making them a leading focus for biotechnology and gene therapy applications. Yet, despite extensive characterization, the structural basis for RNA-guided DNA integration of type I-F3 CASTs has remained elusive. Here, using cryo-EM and functional assays, we report the structural licensing mechanism of the Vibrio cholerae CAST (VchCAST) holo integration complex (HIC). Our structures reveal that sequential conformational licensing events govern selective activation of the integration machinery at the on-target site. Different components, including Cascade-TniQ, TnsC, and TnsA/B, undergo conformational changes upon assembly into the HIC, and the catalytically competent state is stabilized only within the fully assembled complex. Finally, comparison with existing CAST integration complex structures reveals a set of conserved architectural features, suggesting a common molecular basis for RNA-guided transposition across CAST diversity.

biochemistry↗

bioRxiv2026

Programmable DNA integration with New-to-Nature tools using Computational Protein Design

Programmable integration of large DNA cargo ([≥] 2 kb), without inducing double-strand breaks, remains challenging for genome editing technologies. Current approaches have limitations in programmability, depend on co-delivery of multiple components, require multiple enzymatic steps, or have variable on-target editing outcomes. Here, we address this challenge using de novo protein design to create highly active, new-to-nature RNA-guided transposons. Our strategy exploits the modular architecture of CRISPR-associated transposons (CASTs), reconfiguring their conserved transposition machinery to interface with widely adopted Cas9. The resulting system, which we call NovoCAST, simplifies the CAST architecture from eight distinct proteins to four, establishing the simplest CAST described to date. NovoCAST exhibits sharply defined integration profiles, a 500-fold increase in activity relative to the parental PmcCAST, and general programmability. Using structural and biochemical analyses, we confirmed that the designed proteins fold and function as intended. Finally, we demonstrate robust programmable genomic integration in human cells highlighting its broad potential applications in research and therapeutics. Together, these results establish de novo protein design as a powerful strategy for engineering efficient genome-editing systems and for coupling CRISPR-mediated DNA recognition to heterologous functions through de novo designed protein interfaces.

biochemistry↗

bioRxiv2023

Mechanism of target site selection by type V-K CRISPR-associated transposases

Unlike canonical CRISPR-Cas systems that rely on RNA-guided nucleases for target cleavage, CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to facilitate RNA-guided transposition of large genetic payloads. Type V-K CASTs offer several potential upsides for genome engineering, due to their compact size, easy programmability, and unidirectional integration. However, these systems are substantially less accurate than type I-F CASTs, and the molecular basis for this difference has remained elusive. Here we reveal that type V-K CASTs undergo two distinct mobilization pathways with remarkably different specificities: RNA-dependent and RNA-independent transposition. Whereas RNA-dependent transposition relies on Cas12k for accurate target selection, RNA-independent integration events are untargeted and primarily driven by the local availability of TnsC filaments. The cryo-EM structure of the untargeted complex reveals a TnsB-TnsC-TniQ transpososome that encompasses two turns of a TnsC filament and otherwise resembles major architectural aspects of the Cas12k-containing transpososome. Using single-molecule experiments and genome-wide meta-analyses, we found that AT-rich sites are preferred substrates for untargeted transposition and that the TnsB transposase also imparts local specificity, which collectively determine the precise insertion site. Knowledge of these motifs allowed us to direct untargeted transposition events to specific hotspot regions of a plasmid. Finally, by exploiting TnsBs preference for on-target integration and modulating the availability of TnsC, we suppressed RNA-independent transposition events and increased type V-K CAST specificity up to 98.1%, without compromising the efficiency of on-target integration. Collectively, our results reveal the importance of dissecting target site selection mechanisms and highlight new opportunities to leverage CAST systems for accurate, kilobase-scale genome engineering applications.

molecular biology↗

bioRxiv2022

Structures of the holo CRISPR RNA-guided transposon integration complex

CRISPR-associated transposons (CAST) are programmable mobile elements that insert large DNA cargo by an RNA-guided mechanism. Multiple conserved components act in concert at the target site through formation of an integration complex (transpososome). We reconstituted the type V-K CAST transpososome from Scytonema hofmannii (ShCAST) and determined the structure using cryo-EM. Transpososome architecture ensures orientation-specific association: AAA+ regulator TnsC has defined polarity and length, with dedicated interaction interfaces for other CAST components. Interestingly, transposase (TnsB)-TnsC interactions we observe contribute to stimulating TnsCs ATP hydrolysis activity. TnsC deviates from previously observed helical configurations of TnsC, and target DNA does not track with TnsC protomers. Consequently, TnsC makes new, functionally important protein-DNA interactions throughout the transpososome. Finally, two distinct transpososome populations suggests that associations with the CRISPR effector are flexible. These ShCAST transpososome structures significantly enhances our understanding of CAST transposition systems and suggests avenues for improving CAST transposition for precision genome-editing applications.

biophysics↗

bioRxiv2021

CRISPR-Csx28 forms a Cas13b-activated membrane pore required for robust CRISPR-Cas adaptive immunity

Type VI CRISPR-Cas systems use the RNA-guided RNase Cas13 to defend bacteria against viruses, and some of these systems encode putative membrane proteins that have unclear roles in Cas13-mediated defense. Here we show that Csx28, of Type VI-B2 systems, forms membrane pore structures to slow cellular metabolism upon viral infection, and this activity drastically increases anti-viral defense. High- resolution cryo-EM reveals that Csx28 exists unexpectedly as a detergent-encapsulated octameric pore, and we then show these Csx28 pores are membrane localized in vivo. Activation of Csx28 in vivo strictly requires sequence-specific recognition of viral mRNAs by Cas13b, and this activation results in Csx28-mediated membrane depolarization, slowed metabolism, and inhibition of sustained viral infection. Together, our work reveals an unprecedented mechanism by which Csx28 acts as a downstream, Cas13b-activated, effector protein that uses membrane perturbation as an anti-viral defense strategy.

biochemistry↗

bioRxiv2021

Structural basis for target-site selection in RNA-guided DNA transposition systems

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA insertion in one orientation at a fixed distance from a programmable target sequence. We define the mechanism explaining this process by characterizing the transposition regulator, TnsC, from a Type V-K CRISPR-transposase system using cryo-EM. Polymerization of ATP-bound TnsC helical filaments explains how polarity information is passed to the transposase. Our Cryo-EM structure of TniQ-TnsC reveals that TniQ caps the TnsC filament, establishing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, structures with the transition state mimic, ADP{middle dot}AlF3, reveals how TnsC transitions to define the fixed point of insertion. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications. One Sentence SummaryCryo-EM studies reveals the role of the AAA+ regulator TnsC for target-site selection in CRISPR-associated transposition systems.

molecular biology↗

bioRxiv2018

Atomic model of microtubule-bound tau

Tau is a developmentally regulated protein found in axons, whose physiological role is to stabilize and bundle microtubules (MTs). Hyper-phosphorylation of tau is thought to cause its detachment from MTs and subsequent aggregation into pathological fibrils that have been implicated in Alzheimers disease pathogenesis. Despite its known MT binding role, there is no consensus regarding which tau residues are crucial for tau-MT interactions, where on the MT tau binds, and how binding results in MT stabilization. We have used cryo-EM to visualize the interaction of different tau constructs with MTs at high resolution (3.2-4.8 [A]) and used computational approaches to generate atomic models of tau-tubulin interactions. Our work shows that the highly conserved tubulin-binding repeats within tau adopt very similar structures in their interactions with the MT. Each tau repeat binds the MT exterior and adopts an extended structure along the crest of the protofilament (PF), interacting with both - and {beta}-tubulin, thus stabilizing the interface between tubulin dimers. Our structures agree with and explain previous biochemical data concerning the effect of phosphorylation on MT affinity and lead to a model in which tau repeats bind in tandem along a PF, tethering together tubulin dimers and stabilizing longitudinal polymerization interfaces. These structural findings could establish a basis of future treatments aiming at the selective stabilization of tau-MT interactions.

molecular biology↗