Biology subjects

Taylor, D. W.

Publications and source records attributed to Taylor, D. W..

At least 19 recordsLinked to original sources

bioRxiv2026

Asymmetric DNA targeting by RNA-guided TIGR-Tas systems

Tandem interspaced guide RNA (TIGR)-TIGR-associated (Tas) are RNA-guided defense systems, which use a dual-repeat or stem-loop tigRNA to direct a Tas dimer to DNA targets through RNA-DNA heteroduplex formation between both DNA strands. While previous work has shown the basic principles of DNA targeting, how the guide architecture influences target recognition and whether recognition and cleavage are coordinated across the two RNA-DNA heteroduplexes at a target site remain poorly understood. Here, we combine cryo-electron microscopy, biochemistry, and cell-based assays to investigate two TIGR-Tas effectors: the nuclease-lacking Peromyscus leucopus TasA (PlTasA), associated with a stem-loop tigRNA, and the nuclease-active Salicola phage TasH (SpTasH). Cryo-EM structures of PlTasA binary and ternary complexes reveal a dimeric scaffold similar to SpTasH and TaTasR with a distinct stem-loop tigRNA architecture and additional peripheral structural elements. Binding assays using DNA substrates containing local bubbles across the spacer-matching region show equivalent bubbles produced position- and spacer-dependent effects, indicating that target engagement is asymmetric in both PlTasA and SpTasH. Kinetic and cryo-EM analyses of SpTasH further reveal stepwise heteroduplex formation, with a partially engaged intermediate that undergoes substantial conformational rearrangements of the second protomer, preceding a fully paired state poised for catalytic activation. Cleavage of the two DNA strands occurs through a coordinated, slow process and productive cleavage requires stringent surveillance of both heteroduplexes. Together, these findings define a conserved, ordered mechanism of bipartite target recognition and activation shared by TIGR-Tas effectors, expanding our understanding of the molecular principles underlying programmable DNA targeting by TIGR-Tas systems.

biochemistry↗

bioRxiv2026

Structural and kinetic insights into a metagenomics-derived Cas12a with high specificity

CRISPR-Cas12a nucleases provide an attractive alternative to Cas9 due to their compact RNA scaffold, T-rich PAM requirement, and improved target specificity. However, the mechanistic features that govern activity and discrimination across Cas12a orthologs remain incompletely understood. Here, we characterize Cas12a-MG29-1, a highly active and specific nuclease identified through metagenomic mining, using cryogenic electron microscopy, mutational analysis, and kinetic modeling. The Cas12a-MG29-1 structure reveals repositioned flexible loops near the distal end of the R-loop, including reduced engagement of one loop region and additional contacts formed by a second distal loop. Structure-guided mutagenesis and loop-swap experiments indicate that distal R-loop architecture modulates target discrimination in a context-dependent manner. Single-turnover cleavage and stopped-flow measurements show that Cas12a-MG29-1 and AsCas12a form reversible R-loops with similar kinetics but differ in strand cleavage following R-loop formation. Global kinetic modeling demonstrates that Cas12a-MG29-1 exhibits accelerated non-target strand cleavage, shifting kinetic partitioning toward product formation. This faster irreversible commitment provides a mechanistic explanation for enhanced activity and specificity without altering initial target interrogation. Together, these findings identify distal R-loop interactions and catalytic commitment as key determinants of Cas12a function and provide a framework for interpreting and engineering next-generation Cas12a orthologs.

biochemistry↗

bioRxiv2026

Compact adenine base editors to enable therapeutic rescue of Duchenne muscular dystrophy

Adenine base editors (ABEs) have emerged as a powerful gene-editing technology enabling precise and programmable adenine-to-guanine substitutions across the genome. However, their translation into in vivo therapeutics is limited by delivery challenges, as their size exceeds the packaging capacity of adeno-associated virus (AAV). Here, we report the discovery, structural characterization, and engineering of two compact, highly active ABEs built on novel deaminases and Cas9d nucleases, enabling all-in-one, single-vector AAV delivery. Applying these compact ABEs to Duchenne muscular dystrophy (DMD), we demonstrate efficient disruption of conserved splice-acceptor sites at dystrophin exons 45 and 51 in human skeletal muscle cells, enabling therapeutically relevant exon skipping. Together, these ABEs help expand the therapeutic reach of base editing towards diverse tissue types and disease targets.

bioengineering↗

bioRxiv2026

Architecture of a DNA-guided Cas12a

CRISPR/Cas systems have largely been restricted to RNA-guided nucleases. Here, we present the cryo-EM structure of Acidaminococcus sp. Cas12a (AsCas12a) bound to a pseudo-DNA ({Psi}DNA) guide and RNA target, revealing how Cas12a accomplishes DNA-guided RNA recognition. The {Psi}DNA hairpin bridges the recognition and nuclease lobes, mimicking a PAM-proximal duplex and positioning the spacer to allow formation of a canonical RNA-DNA heteroduplex along the REC lobe. This provides a structural framework for its activity and provides a blueprint for future engineering.

biochemistry↗

bioRxiv2025

Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system

Prokaryotic organisms have evolved unique strategies to acquire immunity against the constant threat of bacteriophage (phage) and mobile genetic elements. Hna is a broadly distributed anti-phage immune system that confers resistance against diverse phage by eliciting an abortive infection response. Using a combination of biochemistry, cryo-electron microscopy, and single-molecule fluorescence imaging, we reveal that Hna functions as a 3--5 single-stranded DNA exonuclease that forms an auto-inhibited dimer under physiological ATP concentrations. We observed that Hna autoinhibition can be overcome by incorporation of a phage-encoded single-stranded DNA binding protein (SSB), stimulating unregulated Hna nuclease activity. Furthermore, phage escape mutants encode SSB variants that evade Hna surveillance by adopting higher order stoichiometries with enhanced DNA binding affinity. Our work establishes the molecular basis of Hna-mediated anti-phage activity and provides novel insights into how phage-encoded proteins can directly stimulate a bacterial immune response.

biochemistry↗

bioRxiv2025

A late cytoplasmic surveillance pathway ensures ribosome integrity

Errors in ribosome assembly can produce defective subunits that can lead to aberrant translation events. How faulty ribosomes are recognized and whether they are recognized during assembly or translation remains poorly understood. We utilized a mutation in the ribosomal protein uL16 to track defective 60S subunits through the biogenesis and translation pathways. This mutation deletes a critical loop in the P site and arrests pre- 60S particles during late cytoplasmic maturation. However, simultaneous mutations in the late biogenesis factors Nmd3 and Tif6 bypass this block, releasing defective ribosomes into the translational pool. Cryo-EM and selective ribosome profiling reveal that these ribosomes can form peptide bonds, but stall predominantly at the first few codons. We show that the uL16 mutant ribosomes are detected and targeted for degradation during biogenesis and that they escape degradation if they enter translation. We identify Reh1 as a non-canonical ribosome assembly factor that is required for this surveillance pathway.

molecular biology↗

bioRxiv2025

The CAGE complex: a hollow, megadalton, protein assembly in prokaryotic and eukaryotic microbes

We report the discovery and structure of a previously unknown [~]1 MDa hollow protein assembly, identified during a survey of ciliary complexes from the ciliate Tetrahymena thermophila. By combining mass spectrometry, structure prediction, and cryo-electron microscopy, we define a homotetrameric cage-like complex with a distinctive elliptical architecture and a large internal cavity. A sequence survey revealed several thousand homologs spanning diverse unicellular eukaryotes--including green algae, fungi, amoebozoans, choanoflagellates, and SAR lineages--as well as predominantly gram-negative bacteria, indicating an ancient evolutionary origin and arguing against a eukaryote-specific function. We determined a near-atomic resolution structure of the complex from the slime mold Dictyostelium discoideum, demonstrating conservation of overall architecture and cavity despite low sequence identity. Together, these results establish the CAGE complex (Conserved Assembly in Gram-negative bacteria and Eukaryotes) as a new class of large protein cage broadly distributed across the tree of life. While its biological function remains unknown, its size, architecture, and conservation suggest possible roles in transport or protein/RNA homeostasis.

molecular biology↗

bioRxiv2025

A Cryptic Binding Pocket Regulates the Metal-Dependent Activity of Cas9

Cas9 is a metal-dependent nuclease that has revolutionized gene editing across diverse cells and organisms exhibiting varying ion uptake, metabolism, and concentrations. However, how divalent metals impact its catalytic function, and consequently its editing efficiency in different cells, remains unclear. Here, extensive molecular simulations, Markov State Models, biochemical and NMR experiments, demonstrate that divalent metals - Mg2+, Ca2+, and Co2+ - promote activation of the catalytic HNH domain by binding within a dynamically forming divalent metal binding pocket (DBP) at the HNH-RuvC interface. Mutations in DBP residues disrupt HNH activation and impair the coupled catalytic activity of both nucleases, identifying this cryptic DBP as a key regulator of Cas9s metal-dependent activity. The ionic strength thereby promotes Cas9s conformational activation, while its catalytic activity is metal-specific. These findings are critical to improving the metal-dependent function of Cas9 and its use for genome editing in different cells and organisms.

biophysics↗

bioRxiv2025

Comparative characterization of Cas12f orthologs reveals mechanistic features underlying enhanced genome editing efficiency

Miniature CRISPR-Cas12f nucleases are attractive candidates for therapeutic genome editing owing to their compact size and compatibility with adeno-associated virus (AAV) delivery. However, editing efficiencies in mammalian cells are lower than those of larger systems such as Cas12a and SpCas9. The extensive phylogenetic diversity of Cas12f suggests unexplored mechanistic variation with the potential for optimization. Here, we characterize a naturally occurring Cas12f ortholog discovered through metagenomics, Cas12f-MG119-28, which supports robust genome editing in human cells. Through structural, biochemical, and kinetic analyses, we compare Cas12f-MG119-28 with two recently described orthologs, Oscillibacter sp. Cas12f (OsCas12f) and Ruminiclostridium herbifermentans Cas12f (RhCas12f). These orthologs present divergent architectures and regulatory features governing PAM recognition, gRNA binding, dimerization, and DNA cleavage. Notably, Cas12f-MG119-28 achieves efficient R-loop formation via a stable dimer interface and a naturally optimized guide RNA. These discoveries elucidate key mechanistic determinants of Cas12f activity and may offer a framework for engineering compact genome editors with therapeutic potential.

biochemistry↗

bioRxiv2024

Visualization of a multi-turnover Cas9 after product release

While the most widely used CRISPR-Cas enzyme is the S. pyogenes Cas9 endonuclease (Cas9), it exhibits single-turnover enzyme kinetics which leads to long residence times on product DNA. This blocks access to DNA repair machinery and acts as a major bottleneck during CRISPR-Cas9 gene editing. Although Cas9 can eventually be forcibly removed by extrinsic factors (translocating polymerases, helicases, chromatin modifying complexes, etc), the mechanisms contributing to Cas9 dissociation following cleavage remain poorly understood. Interestingly, its been shown that Cas9 can be more easily dislodged when complexes collide with the PAM-distal region of the Cas9 complex or when the strength of Cas9 interactions in this region are weakened. Here, we employ truncated guide RNAs as a strategy to weaken PAM-distal nucleic acid interactions and still support Cas9 activity. We find that guide truncation promotes much faster Cas9 turnover and used this to capture previously uncharacterized Cas9 reaction states. Kinetics-guided cryo-EM enabled us to enrich for rare, transient states that are often difficult to capture in standard workflows. From a single dataset, we examine the entire conformational landscape of a multi-turnover Cas9, including the first detailed snapshots of Cas9 dissociating from product DNA. We discovered that while the PAM-distal product dissociates from Cas9 following cleavage, tight binding of the PAM-proximal product directly inhibits re-binding of new targets. Our work provides direct evidence as to why Cas9 acts as a single-turnover enzyme and will guide future Cas9 engineering efforts.

biochemistry↗

bioRxiv2024

Mechanism of type II-B CRISPR-Cas enhanced specificity

Cas9 from Streptococcus pyogenes (SpCas9) revolutionized genome editing by enabling programmable DNA cleavage guided by an RNA. However, SpCas9 tolerates mismatches in the DNA-RNA duplex, which can lead to deleterious off-target editing. Here, we reveal that Cas9 from Francisella novicida (FnCas9) possesses a unique structural feature--the REC3 clamp--that underlies its intrinsic high-fidelity DNA targeting. Through kinetic and structural analyses, we show that the REC3 clamp forms critical contacts with the PAM-distal region of the R-loop, thereby imposing a novel checkpoint during enzyme activation. Notably, F. novicida encodes a non-canonical small CRISPR-associated RNA (scaRNA) that enables FnCas9 to repress an endogenous bacterial lipoprotein gene, subverting host immune detection. Structures of FnCas9 with scaRNA illustrate how partial R-loop complementarity hinders REC3 clamp docking and prevents cleavage in favor of transcriptional repression. The REC3 clamp is conserved in type II-B CRISPR-Cas9 systems, pointing to a potential path for engineering precise genome editors or developing novel antibacterial strategies. These findings reveal the dual mechanisms of high specificity and virulence by FnCas9, with broad implications for biotechnology and therapeutic development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/619592v3_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@8806b0org.highwire.dtl.DTLVardef@6f361eorg.highwire.dtl.DTLVardef@149f506org.highwire.dtl.DTLVardef@757f6d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2024

DNA targeting by compact Cas9d and its resurrected ancestor

The type II-A CRISPR effector SpCas9 has gained widespread popularity as an efficient and programmable genome editing tool. However, much remains to be known about novel compact variants that may overcome some limitations of current systems1,2. Recently, alternative CRISPR-Cas systems with highly compact nucleases capable of genome editing in mammalian cells have been discovered through metagenomic analysis of uncultivated microbes, including Cas9d (a type II-D CRISPR-Cas effector)3. Here, we report the cryo-EM structures of a Cas9d nuclease (747 amino acids in length) in multiple functional states, revealing a stepwise process of DNA targeting involving a conformational switch in a REC2 domain insertion. Our structures provide insights into the intricately folded guide RNA which acts as a structural scaffold to anchor small, flexible protein domains and facilitate DNA target recognition. We find that the sgRNA can be truncated by up to [~]25% yet still retain activity in vivo. We also show that despite preferentially targeting an NGG PAM, Cas9d exhibits a unique mechanism for PAM recognition. Finally, we identify the first Cas9d smaller than 800 amino acids exhibiting robust nuclease activity in mammalian cells. Using ancestral sequence reconstruction, we demonstrate that it is possible to generate compact nucleases capable of efficient genome editing by expanding the diversity of Cas9d families. Collectively, our results provide mechanistic insights into the evolution and DNA targeting of diverse type II CRISPR-Cas systems, providing a molecular blueprint for future rational re-engineering of minimal RNA-guided DNA nucleases.

biochemistry↗

bioRxiv2023

An amino acid-resolution interactome for motile cilia illuminates the structure and function of ciliopathy protein complexes

Motile cilia are ancient, evolutionarily conserved organelles whose dysfunction underlies motile ciliopathies, a broad class of human diseases. Motile cilia contain myriad different proteins that assemble into an array of distinct machines, so understanding the interactions and functional hierarchies among them presents an important challenge. Here, we defined the protein interactome of motile axonemes using cross-linking mass spectrometry (XL/MS) in Tetrahymena thermophila. From over 19,000 XLs, we identified 4,757 unique amino acid interactions among 1,143 distinct proteins, providing both macromolecular and atomic-scale insights into diverse ciliary machines, including the Intraflagellar Transport system, axonemal dynein arms, radial spokes, the 96 nm ruler, and microtubule inner proteins, among others. Guided by this dataset, we used vertebrate multiciliated cells to reveal novel functional interactions among several poorly-defined human ciliopathy proteins. The dataset therefore provides a powerful resource for studying the basic biology of an ancient organelle and the molecular etiology of human genetic disease. HighlightsO_LIOver 4,700 distinct amino-acid cross-links reveal the composition, structural organization, and conformational dynamics of proteins in motile ciliary axonemes. C_LIO_LIDense interaction networks are defined for tubulins, Intraflagellar Transport complexes, axonemal dyneins, radial spokes, the CCDC39/40 96nm molecular ruler, and other complexes. C_LIO_LIThese data reveal the placement of multiple adenylate kinases in the central apparatus and radial spokes of motile axonemes, a new microtubule-associated protein complex of CFAP58 and CCDC146, and insights into the activity of ciliopathy protein MAATS1/CFAP91. C_LIO_LIThe data also provide the first known molecular defect resulting from loss of the human ciliopathy protein ENKUR. C_LI

cell biology↗

bioRxiv2023

Lipid droplets as substrates for protein phase separation

Membrane-associated protein phase separation plays critical roles in cell biology, driving essential cellular phenomena from immune signaling to membrane traffic. Importantly, by restricting diffusion to a two-dimensional surface, lipid bilayers can nucleate phase separation at far lower concentrations compared to those required for phase separation in solution. How might other intracellular lipid substrates, such as lipid droplets, contribute to nucleation of phase separation? Distinct from bilayer membranes, lipid droplets consist of a phospholipid monolayer surrounding a core of neutral lipids, and they are energy storage organelles that protect cells from lipotoxicity and oxidative stress. Here, we show that intrinsically disordered proteins can undergo phase separation on the surface of synthetic and cell-derived lipid droplets. Specifically, we find that model disordered domains, FUS LC and LAF1-RGG, separate into protein-rich and protein-depleted phases on the surfaces of lipid droplets. Owing to the hydrophobic nature of interactions between FUS LC proteins, increasing ionic strength drives an increase in its phase separation on droplet surfaces. The opposite is true for LAF1-RGG, owing to the electrostatic nature of its interprotein interactions. In both cases, protein-rich phases on the surfaces of synthetic and cell-derived lipid droplets demonstrate molecular mobility indicative of a liquid-like state. Our results show that lipid droplets can nucleate protein condensates, suggesting that protein phase separation could be key in organizing biological processes involving lipid droplets.

biophysics↗

bioRxiv2023

Type III-B CRISPR-Cas signaling-based cascade of proteolytic cleavages

Type III CRISPR-Cas systems provide a sequence-specific adaptive immune response that protects prokaryotic hosts against viruses and other foreign genetic invaders. These crRNA-guided Cas effector complexes bind and cleave complementary RNA targets. Specific target binding stimulates the Cas10 subunit to generate cyclic oligoadenylate (cOA) signaling molecules, that in turn allosterically activate proteins carrying cognate sensory domains: CARF or SAVED. Here, we characterize an elaborate set of genes associated with the type III-B CRISPR-Cas system from Haliangium ochraceum, which includes a signal transduction module of a CBASS defense system with two caspase-like proteases, SAVED-CHAT and PCaspase (Prokaryotic Caspase). We show that binding of a 3-nucleotide cOA (cA3) to the SAVED domain of SAVED-CHAT induces its oligomerization into long filaments that activate the proteolytic activity of the CHAT domain. Surprisingly, we find that activated SAVED-CHAT specifically cleaves and activates the second protease, PCaspase. In turn, activated PCaspase cleaves a multitude of other proteins, including a putative sigma factor and a PCaspase-inhibitor. We expressed the type III-B system and its associated genes in E. coli and observed a strong abortive phenotype when offering a complementary target RNA, but only in the presence of both SAVED-CHAT and PCaspase. Together, our findings show an intriguing cascade of proteolytic activities (conceptually similar to eukaryotic caspases) in this bacterial immune system that reveals yet another strategy to effectively defend against mobile genetic elements.

biochemistry↗

bioRxiv2023

Unraveling the mechanisms of PAMless DNA interrogation by SpRY Cas9

CRISPR-Cas9 is a powerful tool for genome editing, but the strict requirement for an "NGG" protospacer-adjacent motif (PAM) sequence immediately adjacent to the DNA target limits the number of editable genes. To overcome the PAM requirement, a recently developed Cas9 variant, called SpRY-Cas9 was engineered to be "PAMless" (1, 2). However, the molecular mechanisms of how SpRY can recognize all potential PAM sequences and still accurately identify DNA targets have not been investigated. Here, we combined enzyme kinetics, cryo-EM, and single-molecule imaging to determine how SpRY interrogates DNA and recognizes target sites for cleavage. Divergent PAM sequences can be accommodated through conformational flexibility within the PAM-interacting region of SpRY, which facilitates tight binding to off-target DNA sequences. Once SpRY correctly identifies a target site, nuclease activation occurs [~]1,000-fold slower than for Streptococcus pyogenes Cas9, enabling us to directly visualize multiple on-pathway intermediate states. Insights gained from our intermediate structures prompted rationally designed mutants with improved DNA cleavage efficiency. Our findings shed light on the molecular mechanisms of PAMless genome editing with SpRY and provide a framework for the design of future genome editing tools with improved versatility, precision, and efficiency.

biochemistry↗

bioRxiv2023

Structural basis of Cas12a R-loop propagation on pathway to DNA cleavage

Cas12a is a CRISPR RNA-guided nuclease that engages target DNA through protein-DNA and RNA-DNA contacts. Initial PAM binding by Cas12a leads to formation of a 20bp R-loop between the complementary crRNA guide and target strand. Following specificity-determining R-loop formation, both DNA strands undergo RuvC-mediated cleavage. Current structures of Cas12a bound to its target only show the R-loop after formation, leaving an important gap in knowledge as to how Cas12a accommodates the extending R-loop and coordinates R-loop formation with nuclease activation. Here, we use cryo-EM to capture a series of kinetically trapped Cas12a R-loop intermediates and observe how Cas12a delivers each DNA strand into the RuvC active site. We show that Cas12a first interrogates target DNA via a 5bp seed, followed by dramatic Rec domain conformational flexibility to accommodate R-loop extension. Only during formation of the final R-loop base pairs do the Rec and BH domains engage in the majority of contacts with the R-loop. R-loop completion leads the nontarget strand to displace the RuvC lid and kink into the active site via a base stacking interaction. Following nontarget strand cleavage, we observe substantial Rec2 and Nuc domain dynamics as the TS is brought to the RuvC active site. Our kinetics-guided structural snapshots provide a comprehensive model describing Cas12a DNA targeting and highlight mechanistic differences between Cas12a and Cas9.

biochemistry↗

bioRxiv2022

Does AlphaFold2 model proteins' intracellular conformations? An experimental test using cross-linking mass spectrometry of endogenous ciliary proteins

A major goal in structural biology is to understand protein assemblies in their biologically relevant states. Here, we investigate whether AlphaFold2 structure predictions match native protein conformations. We chemically cross-linked proteins in situ within intact Tetrahymena thermophila cilia and native ciliary extracts and identified 1,225 intramolecular cross-links within the 100 best-sampled proteins to provide a benchmark of distance restraints obeyed by proteins in their native assemblies. The corresponding AlphaFold2 structure predictions were highly concordant, positioning 86.2% of cross-linked residues within C-to-C distances of 30 [A], consistent with the known cross-linker length. 43% of the proteins showed no violations. Most inconsistencies occurred in low-confidence regions or between domains of the structure prediction. For basal body protein BBC118, cross-links combined with the predicted structure revealed domain packing satisfying both data. Overall, AlphaFold2 predicted biological structures with low predicted aligned error corresponded to more correct native structures. However, we observe cases where rigid body domains are oriented incorrectly, suggesting that combining structure prediction with experimental information will better reveal biologically relevant conformations.

bioinformatics↗