Biology subjects

Jones, S. K.

Publications and source records attributed to Jones, S. K..

13 recordsLinked to original sources

bioRxiv2026

Cas12a cleavage and trimming kinetics reveal mismatches as a tool to steer editing

Gene knockouts by CRISPR-Cas nucleases rely on targeted DNA cleavage and error-prone DNA repair: end-joining pathways can introduce insertions and deletions that assist in disrupting the coding sequence. However, only a fraction of edits achieves this, and an unfavorable array of repair outcomes typically requires switching to another editing technology. Key factors that influence repair are the types and lengths of DNA ends following cleavage. Here, we investigated Cas12a's ability to produce different ends and if they can be used to redistribute editing outcomes. We determined the sites and rates of target cleavage by Cas12a in vitro by combining kinetic modeling with nucleotide-resolution assays. Here, we show that trimming - repeated cleavage of an already cut target - occurs about 4x faster than initial cleavage; it also presents alternative DNA end structures for cellular repair. We also introduced specific mismatches to the gRNA: Cas12a maintained fast target cleavage, but changed where the target was cleaved and how quickly it was trimmed, compared to matched gRNA. We exploited the differences in cleavage kinetics between matched and mismatched gRNAs to develop reprogrammed gRNAs, i.e. rpgRNAs. Intentionally-mismatched rpgRNAs retained the high editing efficiency observed with traditional gRNAs. However, they shifted editing between in-frame and out-of-frame outcomes to enhance gene knockout success across genes. Reprogrammed gRNAs offer an efficient way to steer editing toward such preferred outcomes, while retaining the simplicity of gene editing with CRISPR-Cas nucleases.

biochemistry↗

bioRxiv2026

Targeted DNA nicking enables efficient, single-step and counterselection-free editing of bacteriophage genomes

Genetic manipulation of bacteriophages is essential for interrogating phage biology and advancing antimicrobial therapies. However, current genome editing approaches can be inefficient, require multiple steps, or drastically reduce phage titers. Here, we show that targeted DNA nicking enables template-mediated editing of phage genomes in one step without reducing phage titers. Using T7 phage, we show that Cas9-mediated nicking achieved up to 100% recombination across multiple loci, including substitutions and deletions of up to 200 bp and insertions of up to 500 bp, all while preserving phage titers. Editing in T7 was RecA-independent and extended to other phages. Leveraging high titers, we engineered a T7 library of over 440,000 tail-fiber mutants, with isolated mutants restoring infection of two LPS-deficient Escherichia coli hosts by shifting recognition to core LPS components. Overall, DNA nicking is a simple and distinct editing strategy that can advance phage genome engineering, genetic interrogation, and antimicrobial development.

synthetic biology↗

bioRxiv2026

Supercoiling twists Cas9 off-target discrimination when nicking and cleaving

Programmed with an RNA guide, Cas9 nuclease directs double-strand DNA cleavage via its two nuclease domains. However, Cas9 sometimes falsely identifies DNA targets by binding and cleaving DNA that does not match its PAM and guide RNA sequence requirements. Cas9s specificity is often affected by DNA topology, as DNA negative supercoiling can increase off-target activity while positive supercoiling can even prevent on-target activity. Such dramatic DNA topological changes routinely occur in cells as a result of transcription and replication, making Cas9s specificity a challenge for gene editing. To determine how Cas9 imparts its specificity across sequences and topologies, we directly mapped kinetic cleavage rates and sites for thousands of relaxed and negatively-supercoiled target sequences via NucleaSeq. We find that: Negative supercoiling can accelerate off-target cleavage a thousand-fold, and shift cleavage sites by two nucleotides. Guide-target mispairs differently impact RuvC and HNH domains, which can lead to topology-dependent nicking by Cas9. Finally, we predict these variations in Cas9 cleavage activity with a biophysical model that accounts for DNA topological state. These efforts expose Cas9s strand-specific off-target cleavage activity and can improve off-target identification for more predictable and safer gene editing. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=94 SRC="FIGDIR/small/715559v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c1d4c5org.highwire.dtl.DTLVardef@77d891org.highwire.dtl.DTLVardef@2b3073org.highwire.dtl.DTLVardef@179d442_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2025

A mechanistic free-energy model explains and predicts sequence- and context-dependent CRISPR-Cas9 activity

Accurate prediction of CRISPR-based gene editing remains challenging since existing models often fail to generalize across experimental and cellular contexts. We introduce CRISPRzip, a mechanistic kinetic model that quantitatively links nucleotide sequence and environmental conditions to target interrogation. The model describes R-loop formation as movement through a sequence-dependent free-energy landscape, combining nearest-neighbor nucleic-acid energetics with protein-mediated contributions inferred from high-throughput binding and cleavage kinetics. Applying CRISPRzip to SpCas9, we predict the activity across diverse DNA targets and guide RNAs, and validate with independent singlemolecule FRET and torque spectroscopy experiments. By explicitly incorporating Cas9 concentration and DNA superhelicity, the framework predicts heterogeneous, context-dependent editing outcomes and rationalizes how physical constraints modulate cleavage dynamics. Our results provide a transferable, physics-based foundation that unifies mechanistic insight and predictive power, enabling robust characterization of CRISPR effectors and prediction of their activity across environmental contexts.

biophysics↗

bioRxiv2024

Global review of meta-analyses reveals key data gaps in agricultural impact studies on biodiversity in croplands

AimAgriculture depends heavily on biodiversity, yet unsustainable management practices continue to affect a wide range of organisms and ecosystems at unprecedented levels worldwide. Addressing the global challenge of biodiversity loss requires access to consolidated knowledge across management practices, spatial levels, and taxonomic groups. LocationGlobal Time period1994 to 2022 Major taxa studiedAnimals, microorganisms, plants. MethodsWe conducted a comprehensive literature review synthesising data from all meta-analyses about the impacts of agricultural management practices on biodiversity in croplands, covering field, farm, and landscape levels. From 200 retained meta-analyses, we extracted 1,885 mean effect sizes (from 69,850 comparisons between a control and treatment) assessing the impact of management practices on biodiversity, alongside characterising over 9,000 primary papers. ResultsSeven high-income countries, notably the USA, China, and Brazil dominate agricultural impact studies with fertiliser use, phytosanitary interventions and crop diversification receiving widespread attention. The focus on individual practices overshadows research at the farm and landscape level. Taxonomically, Animalia, especially arthropods, are heavily studied while taxa such as annelids and plants receive comparatively less attention. Effect sizes are predominantly calculated from averaged abundance data. Significant gaps persist in terms of studies on the effects of agricultural interventions on specific taxonomic groups (e.g. annelids, mammals) and studies analysing functional traits. Main conclusionsOur study highlights the importance of analysing the effects of combined practices to accurately reflect real-world farming contexts. While abundance metrics are common, reflecting several biodiversity facets and adopting a more balanced research approach across taxa are crucial for understanding biodiversity responses to agricultural changes and informing conservation strategies. Given the unbalanced evidence on impacts of agricultural practices on biodiversity, caution is required when utilising meta-analytical findings for informing public policies or integrating them into global assessment models like life-cycle assessments or global flux models.

ecology↗

bioRxiv2023

Learning and memory deficits produced by aspartame are heritable via the paternal lineage

Environmental exposures produce heritable traits that can linger in the population for one or two generations. Millions of individuals consume substances such as artificial sweeteners daily that are declared safe by regulatory agencies without evaluation of their potential heritable effects. We show that consumption of aspartame, an FDA-approved artificial sweetener, daily for up to 16-weeks at doses equivalent to only 7-15% of the FDA recommended maximum daily intake value (equivalent to 2-4 small, 8 oz diet soda drinks per day) produces significant spatial learning and memory deficits in mice. Moreover, the cognitive deficits are transmitted to male and female descendants along the paternal lineage suggesting that aspartames adverse cognitive effects are heritable, and that they are more pervasive than current estimates, which consider effects in the directly exposed individuals only. Traditionally, deleterious environmental exposures of pregnant and nursing women are viewed as risk factors for the health of future generations. Environmental exposures of men are not considered to pose similar risks. Our findings suggest that environmental exposures of men can produce adverse impact on mental health of future generations and demonstrate the need for considering heritable effects via the paternal lineage as part of the regulatory evaluations of artificial sweeteners.

neuroscience↗

bioRxiv2023

Advancing Understanding of DNA-BfiI Restriction Endonuclease Cis and Trans Interactions through smFRET Technology

Monitoring of DNA-protein interactions is essential in understanding many biological processes. Proteins must find their target site on a DNA molecule to perform their function, and the mechanisms for target search differ across proteins. Revealing temporal interactions with two target sites, both in Cis and in Trans, is crucial in target search mechanisms studies. Here, we present two single-molecule Forster resonance energy transfer (smFRET)-based assays to study BfiI-DNA interactions. The first assay, smFRET-based DNA looping assay, detects both "Phi" and "U"-shaped DNA looping events. We modified it to only allow in Trans BfiI-target DNA interactions to improve specificity and reduce limitations in the observation time. Our TIRF microscopy measurements directly observe the on- and off-target binding events and characterize BfiI binding events. Our results show that BfiI binding events last longer on target sites and that the BfiI rarely changes conformations during binding. This newly developed assay could be useful for other two-targets-binding DNA-interacting proteins and could be employed for dsDNA substrate BfiI-PAINT, which is useful for DNA stretch-assays and other super-resolution fluorescence microscopy studies.

biophysics↗

bioRxiv2022

Biosphere functional integrity for people and Planet

Defining a safe and just biosphere space requires a synthetic scaleable measure of biosphere functional integrity to secure Natures Contributions to People (NCP). Using a systematic review of 153 peer-reviewed studies we estimated the minimum level of functional integrity needed to secure multiple critical NCP, including pollination, pest and disease control, water quality regulation, soil protection, recreation and natural hazards mitigation in human-modified landscapes. We characterise functional integrity by the quantity, quality and spatial configuration of (semi-)natural habitat within any landscape. We find that at least 20-25% of structurally complex and biologically diverse (semi-)natural habitat in each 1 km2 of land area is needed to maintain the supply of multiple NCP simultaneously. Exact quantity, quality and spatial configuration required is dependent on local context, and may differ for individual NCP. Today, about 50-60% of human-modified lands have less than 10% and 20% (semi-)natural habitat per 1 km2 respectively. These areas require immediate attention to regenerate functional integrity in order to secure ecological functioning in those landscapes.

ecology↗

bioRxiv2020

The Polyadenosine RNA Binding Protein ZC3H14 is Required in Mice for Proper Dendritic Spine Density

ZC3H14 (Zinc finger CysCysCysHis domain-containing protein 14), an evolutionarily conserved member of a class of tandem zinc finger (CCCH) polyadenosine (polyA) RNA binding proteins, is associated with a form of heritable, nonsyndromic autosomal recessive intellectual disability. Previous studies of a loss of function mouse model, Zc3h14{Delta}ex13/{Delta}ex13, provide evidence that ZC3H14 is essential for proper brain function, specifically for working memory. To expand on these findings, we analyzed the dendritic spines of hippocampal neurons from Zc3h14{Delta}ex13/{Delta}ex13 mice. These studies reveal that loss of ZC3H14 does not affect dendritic spine density in either CA1 pyramidal neurons or dentate gyrus granule cells in the hippocampus. However, overexpression of ZC3H14 in cultured hippocampal neurons increases the overall density of spines. We next performed biochemical analyses of synaptosomes prepared from whole wild-type and Zc3h14{Delta}ex13/{Delta}ex13 mouse brains to determine if there are changes in steady state levels of postsynaptic proteins upon the loss of ZC3H14. We found that ZC3H14 is present within synaptosomes and that a crucial postsynaptic protein, CaMKII, is significantly increased in these synaptosomal fractions upon loss of ZC3H14. Together, these results demonstrate that ZC3H14 localizes to synapses, that its levels influence dendritic spine morphology, and that its loss dysregulates synaptic CaMKII levels, suggesting a potential role for ZC3H14 in synaptic function and plasticity.

neuroscience↗

bioRxiv2020

A mechanistic model improves off-target predictions and reveals the physical basis of SpCas9 fidelity

The S. pyogenes (Sp) Cas9 endonuclease is an important gene-editing tool. SpCas9 is directed to target sites via a single guide RNA (sgRNA). However, SpCas9 also binds and cleaves genomic off-target sites that are partially matched to the sgRNA. Here, we report a microscopic kinetic model that simultaneously captures binding and cleavage dynamics for SpCas9 and Sp-dCas9 in free-energy terms. This model not only outperforms state-of-the-art off-target prediction tools, but also details how Sp-Cas9s structure-function relation manifests itself in binding and cleavage dynamics. Based on the biophysical parameters we extract, our model predicts SpCas9s open, intermediate, and closed complex configurations and indicates that R-loop progression is tightly coupled with structural changes in the targeting complex. We show that SpCas9 targeting kinetics are tuned for extended sequence specificity while maintaining on-target efficiency. Our extensible approach can characterize any CRISPR-Cas nuclease - benchmarking natural and future high-fidelity variants against SpCas9; elucidating determinants of CRISPR fidelity; and revealing pathways to increased specificity and efficiency in engineered systems.

biophysics↗

bioRxiv2019

Massively parallel kinetic profiling of natural and engineered CRISPR nucleases

Engineered Streptococcus pyogenes (Sp) Cas9s and Acidaminococcus sp. (As) Cas12a (formerly Cpf1) improve cleavage specificity in human cells. However, the fidelity, enzymatic mechanisms, and cleavage products of emerging CRISPR nucleases have not been profiled systematically across partially mispaired off-target DNA sequences. Here, we describe NucleaSeq-- nuclease digestion and deep sequencing--a massively parallel platform that measures cleavage kinetics and captures the time-resolved identities of cleaved products for more than ten thousand DNA targets that include mismatches, insertions, and deletions relative to the guide RNA. The binding specificity of each enzyme is measured on the same DNA library via the chip-hybridized association mapping platform (CHAMP). Using this integrated cleavage and binding platform, we profile four SpCas9 variants and AsCas12a. Engineered Cas9s retain wtCas9-like off-target binding but increase cleavage specificity; Cas9-HF1 shows the most dramatic increase in cleavage specificity. Surprisingly, wtCas12a--reported as a more specific nuclease in cells--has cleavage specificity similar to wtCas9 in vitro. Initial cleavage position and subsequent end-trimming vary across nucleases, guide RNA sequences, and position and base identity of mispairs in target DNAs. Using these large datasets, we develop a biophysical model that reveals mechanistic insights into off-target cleavage activities by these nucleases. More broadly, NucleaSeq enables rapid, quantitative, and systematic comparison of the specificities and cleavage products of engineered and natural nucleases.

molecular biology↗

bioRxiv2018

Error-correcting DNA barcodes for high-throughput sequencing

Many large-scale high-throughput experiments use DNA barcodes--short DNA sequences prepended to DNA libraries--for identification of individuals in pooled biomolecule populations. However, DNA synthesis and sequencing errors confound the correct interpretation of observed barcodes and can lead to significant data loss or spurious results. Widely-used error-correcting codes borrowed from computer science (e.g., Hamming and Levenshtein codes) do not properly account for insertions and deletions in DNA barcodes, even though deletions are the most common type of synthesis error. Here, we present and experimentally validate FREE (Filled/truncated Right End Edit) barcodes, which correct substitution, insertion, and deletion errors, even when these errors alter the barcode length. FREE barcodes are designed with experimental considerations in mind, including balanced GC content, minimal homopolymer runs, and reduced internal hairpin propensity. We generate and include lists of barcodes with different lengths and error-correction levels that may be useful in diverse high-throughput applications, including >106 single-error correcting 16-mers that strike a balance between decoding accuracy, barcode length, and library size. Moreover, concatenating two or more FREE codes into a single barcode increases the available barcode space combinatorially, generating lists with > 1015 error-correcting barcodes. The included software for creating barcode libraries and decoding sequenced barcodes is efficient and designed to be user-friendly for the general biology community.\n\nSIGNIFICANCE STATEMENTModern high-throughput biological assays study pooled populations of individual members by labeling each member with a unique DNA sequence called a \"barcode.\" DNA barcodes are frequently corrupted by DNA synthesis and sequencing errors, leading to significant data loss and incorrect data interpretation. Here, we describe a novel error-correction strategy to improve the efficiency and statistical power of DNA barcodes. To our knowledge, this is the first report of an error-correcting method that accurately handles insertions and deletions in DNA barcodes, the most common type of error encountered during DNA synthesis and sequencing, resulting in order-of-magnitude increases in accuracy, efficiency, and signal-to-noise. The accompanying software package makes deployment of these barcodes effortless for the broader experimental scientist community.

molecular biology↗

bioRxiv2017

A memory element imposes epigenetic behavior on intrinsically labile RNAi-induced heterochromatin spread.

ABTRACTThe heterochromatin spreading reaction is a central contributor to the formation of gene-repressive structures, which are re-established with high fidelity following replication. The high fidelity of this process is not obviously encoded in the primary spreading reaction. To resolve origins of stable inheritance of repression, we probed the intrinsic fidelity of spreading events in fission yeast using a system that quantitatively describes the spreading reaction in live single cells. We show that spreading from RNAi-nucleated elements is stochastic, multimodal, and fluctuates dynamically across time. In contrast, a second form of spreading, nucleated by the cis-acting element REIII, is deterministic, has high memory capacity and acts as the source of locus fidelity. REIII enables fidelity in part by endowing the locus with resistance to perturbations. Together, our results suggest that epigenetic capacity may not be intrinsically encoded in the spreading reaction, but rather requires collaboration with specialized memory elements.

genetics↗