Biology subjects

Dangerfield, T. L.

Publications and source records attributed to Dangerfield, T. L..

9 recordsLinked to original sources

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

Transient signaling of free ADP-ribose monitored with an intracellular biosensor

We have developed a biosensor enabling the dynamic, compartmentalized, and longitudinal measurements of intracellular ADP-ribose (ADPR) in live cells. Free ADPR is a critical signaling metabolite derived from nicotinamide adenine dinucleotide (NAD+). As an agonist for Transient Receptor Potential Melastatin 2 (TRPM2), ADPR levels can regulate immune responses during infection, as well as nociception and adjustment of core body temperature. The study of ADPR signaling has been limited, however, by a lack of methods to measure this metabolite in situ. Using the biosensor and its paired non-responsive control, we determine that intracellular ADPR accumulation was transient and tunable. We found that basal concentrations were in the nanomolar range and could be stimulated [~]30-fold to activate TRPM2. We identified that TRPM2 activation, measured by calcium influx, required an intracellular ADPR threshold concentration between 2 - 4 {micro}M at physiological temperature. We observed that the timing of the ADPR rise coincided with TRPM2 activation, thus providing support for ADPR fluctuations being a critically regulated aspect for channel activation. Notably, transient fluctuations of ADPR were not accurately reflected by measurements of intracellular NAD+ loss or calcium levels. Significance StatementWe have developed a unique real-time biosensor for free ADP-ribose that is tuned to physiological concentrations and capable of intracellular measurements in individual cells. Using a calibrated system we determined that concentrations and timing of induced intracellular ADPR aligned with the thermosensitive TRPM2 activity. The data support ADPR as a critical component whose intracellular levels are regulated to control TRPM2 channel opening in cells.

biochemistry↗

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

Single-turnover kinetic analysis of non-LTR retrotransposition defines the mechanism and rate constants governing each step

Site specific retrotransposon-mediated gene therapy has the potential to revolutionize medicine by allowing insertion of large gene cargos. Despite decades of effort, the reaction sequence remains to be fully elucidated limiting the ability to engineer improved activity for gene insertion. Here we provide a kinetic/mechanistic framework for R2 non-LTR retrotransposition. Single turnover measurements and global data fitting defined the rate constants for each step in the pathway involving 1st-strand DNA cleavage to provide a DNA primer, reverse transcription to copy the RNA, 2nd-strand DNA cleavage to provide the second primer, and 2nd-strand synthesis to make duplex cDNA. Sequence analysis of the cDNA confirms accurate replication of the 1400 nt RNA used in this study. This represents the only complete analysis of the reaction sequence and first observation of 2nd-strand synthesis in vitro. We provide a kinetic framework to understand non-LTR retrotransposition, which provide a basis to engineer improved activity.

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

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↗

bioRxiv2022

Assembly of multi-subunit fusion proteins into the RNA-targeting type III-D CRISPR-Cas effector complex

CRISPR (Clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems are a type of adaptive immune response in bacteria and archaea that utilize crRNA (CRISPR RNA)-guided effector complexes to target complementary RNA or DNA for destruction. The prototypical type III-A and III-B CRISPR-Cas systems utilize multi-subunit effector complexes composed of individual proteins to cleave ssRNA targets at 6-nt intervals, as well as non-specifically degrading ssDNA and activating cyclic oligoadenylate (cOA) synthesis. Recent studies have shown that type III systems can contain subunit fusions yet maintain canonical type III RNA-targeting capabilities. To understand how a multi-subunit fusion effector functions, we determine structures of a variant type III-D effector and biochemically characterize how it cleaves RNA targets. These findings provide insights into how multi-subunit fusion proteins are tethered together and assemble into an active and programmable RNA endonuclease, how the effector utilizes a novel mechanism for target RNA seeding, and the structural basis for the evolution of type III effector complexes. Furthermore, our results provide a blueprint for fusing subunits in class 1 effectors for design of user-defined effector complexes with disparate activities. Important noteWhile this manuscript was in preparation, a manuscript describing the structure of the type III-E effector was published1. We reference these important findings; however, a careful comparison of the structures will follow once the coordinates have been released by the PDB.

biochemistry↗

bioRxiv2021

Conformational dynamics during misincorporation and mismatch extension defined using a DNA polymerase with a fluorescent artificial amino acid

High-fidelity DNA polymerases select the correct nucleotide over the structurally similar incorrect nucleotides with extremely high specificity while maintaining fast rates of incorporation. Previous analysis revealed the conformational dynamics and complete kinetic pathway governing correct nucleotide incorporation using a high-fidelity DNA polymerase variant containing a fluorescent unnatural amino acid. Here we extend this analysis to investigate the kinetics of nucleotide misincorporation and mismatch extension. We report the specificity constants for all possible misincorporations and characterize the conformational dynamics of the enzyme during misincorporation and mismatch extension. We present free energy profiles based on the kinetic measurements and discuss the effect of different steps on specificity. During mismatch incorporation and subsequent extension (with the correct nucleotide), the rates of the conformational change and chemistry are both greatly reduced. The nucleotide dissociation rate, however, increases to greatly exceed the rate of chemistry. To investigate the structural basis for discrimination against mismatched nucleotides, we performed all atom molecular dynamics simulations on complexes with either the correct or mismatched nucleotide bound at the polymerase active site. We show that the closed form of the enzyme with a mismatch bound is greatly destabilized due to weaker interactions with active site residues, non-ideal base pairing, and a large increase in the distance from the 3-OH group of the primer strand to the -phosphate of the incoming nucleotide, explaining the reduced rates of misincorporation. The observed kinetic and structural mechanisms governing nucleotide misincorporation reveal the general principles likely applicable to other high fidelity DNA polymerases.

biochemistry↗

bioRxiv2020

Remdesivir is a delayed translocation inhibitor of SARS CoV-2 replication in vitro

Remdesivir is a nucleoside analog approved by the FDA for treatment of COVID-19. Here, we present a 3.9-[A]-resolution cryoEM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of three copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of three bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

biochemistry↗