Biology subjects

Walker, J. A.

Publications and source records attributed to Walker, J. A..

At least 19 recordsLinked to original sources

bioRxiv2026

Sex-specific effects of exercise on motor coordination and extended basal ganglia physiology.

Exercise broadly affects the basal ganglia, brain structures involved in motor coordination. Exercise-induced changes in these regions can improve pathological conditions such as Parkinsons disease and substance use disorders. Importantly, biological sex is a significant factor in the effects of exercise and in the presentation of these basal ganglia-related conditions. Here, we find surprising sex differences in exercises influence over motor coordination and neural activity across three extended basal ganglia structures: dorsomedial striatum cholinergic interneurons (CINs), substantia nigra pars compacta (SNc) dopaminergic neurons, and caudal pedunculopontine nucleus (PPN) cholinergic neurons. Using voluntary wheel running, accelerating rotarod, ex vivo electrophysiology, and morphological reconstructions, we found that exercise enhances motor coordination, increases SNc excitability, and strengthens excitatory input onto the PPN selectively in female mice. By contrast, exercise increases spontaneous firing rate and reduces dendritic complexity selectively in male CINs. These data reveal sex-specific exercise effects correlated across behavioral and cellular levels. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=192 SRC="FIGDIR/small/720719v1_ufig1.gif" ALT="Figure 1"> View larger version (57K): org.highwire.dtl.DTLVardef@d0f755org.highwire.dtl.DTLVardef@11e2335org.highwire.dtl.DTLVardef@199a591org.highwire.dtl.DTLVardef@44e4d9_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIOne week of exercise enhances motor coordination in females but not males C_LIO_LIExercise increases SNc excitability and excitatory input onto the PPN in females C_LIO_LIExercise increases dorsomedial striatal cholinergic neurons activity in males C_LIO_LIBaseline sex differences in morphology of striatal cholinergic and SNc neurons C_LI

neuroscience↗

bioRxiv2026

Metaplastic sleep regulation in Drosophila determined by microscale circadian neural dynamics

The biophysical mechanisms by which circadian clock neurons integrate temporal coding signals to regulate sleep remain elusive. Here, using Drosophila, we identify Rabphilin (Rph) in DN1p clock neurons as a key stabilizer of the metaplasticity setpoint governing circadian regulation of sleep. Rph protein levels are elevated at night relative to daytime and modulate stochastic process of DN1p membrane potential dynamics linked to variability in synaptic activity at connections between DN1p neurons and their downstream postsynaptic partners. We find that Rph acts as a bidirectional regulator of synaptic plasticity thresholds. Under dim nocturnal light stimulation, Rph knockdown permits synaptic potentiation, whereas synthesized Rph introduction induces synaptic depression. In contrast, under optogenetic manipulation mimicking daytime spiking in DN1p neurons, these effects are reversed. We further show that spike-timing-dependent plasticity emerges when postsynaptic spiking is engaged, with nocturnal dim light conditions determining the direction of plasticity. Together, these findings establish a mechanistic link between microscale circadian neural dynamics and hierarchical metaplastic regulation, demonstrating how circadian regulation of sleep dynamically balances stability and adaptive flexibility through circadian setpoints and environmental nocturnal light interactions. Significance StatementWe show that circadian metaplasticity regulates sleep through membrane potential dynamics. Circadian clock neurons implement flexible metaplasticity, whereby the direction can be determined by internal circadian setpoints and interactions with nocturnal environmental light. This mechanism engages spike-timing-dependent plasticity to determine plasticity polarity. Our findings identify membrane potential dynamics as a computational substrate for physiological state control, linking molecular mechanisms to circuit-level circadian regulation of sleep. Together, they reframe sleep regulation as an active metaplastic process that hierarchically integrates microscale circadian neural dynamics to optimize circuit function.

neuroscience↗

bioRxiv2025

Island syndrome in the critically endangered Lord Howe Island cockroach Panesthia lata

Following island colonisation, organisms experience a unique array of selective pressures, giving rise to a somewhat predictable suite of morphological, demographic and ecological adaptations known as the "island syndrome". Studies of the island syndrome have provided valuable insights into processes of speciation, community assembly, adaptive radiation and ecological release, alongside many others. However, to date, behavioural aspects of island adaptation have comparatively received little scientific attention, especially among invertebrates. In this study we examined the agonistic, courtship and aggregation behaviour of the endangered Lord Howe Island cockroach Panesthia lata, and compared these to its Australian sister species Panesthia cribrata. Behavioural assays revealed that while courtship behaviour was relatively stable across the two species, there was a significantly lower incidence of male agonism in P. lata. In concordence, analyses of nuclear single-nucleotide polymorphisms showed that P. lata forms large aggregations of unrelated individuals, unlike most Panesthia species, which maintain stable family groups. These results align with previous findings of relaxed intraspecific aggression in island mammals and reptiles, providing novel evidence of behavioural island syndrome in an invertebrate. We also found that courtship behaviour did not vary when P. lata interacted with conspecifics from the same or different populations, suggesting that individuals from different populations will readily interbreed. This is a promising outcome for the conservation of this critically endangered species, which currently spans a fragmentary range consisting of small, insular populations.

evolutionary biology↗

bioRxiv2025

Drosophila Modeling of Insomnia-Associated Genes Reveals Diverse Underlying Sleep Phenotypes

Insomnia is a prevalent sleep disorder with highly heterogenous manifestations. While data-driven approaches to insomnia subtyping have revealed potential differences between proposed insomnia subtypes and their impacts on overall health, little is known about the genetic factors that underly and differentiate these potential insomnia subtypes. We utilize a human-genetics driven approach to Drosophila modeling to identify the range of sleep traits regulated by insomnia-associated genes. Modeling pan-neuronal loss of Drosophila orthologs of a set of insomnia genes reveals a broad range of sleep phenotypes. Through systematic characterization of traits related to sleep quantity, timing, and quality, we identify genetic factors that co-regulate aspects of the insomnia-associated phenotypic landscape. Out of the 75 insomnia-associated genes identified, only 1/3 had at least one Drosophila ortholog that regulated overall sleep quantity. In contrast, 1/3 of the insomnia-associated genes had at least one Drosophila ortholog that regulated either sleep timing or sleep quality, without impacting sleep quantity. Together this work, in Drosophila, provides support for a genetic influence on the differences between insomnia subtypes.

neuroscience↗

bioRxiv2025

Drosophila Modeling Identifies Increased Sleep as a Link Between Insomnia and Cardiovascular Disease

Insomnia is a common sleep disorder associated with negative long-term health outcomes, including cardiovascular disease (CVD). We selected 16 genes from 13 insomnia- and CVD- associated genetic loci and disrupted Drosophila melanogaster orthologs in neuronal or cardiac tissue to characterize their roles in regulating sleep and cardiac physiology. Neuronal disruption of four orthologs (APOB, FUR, CYP17A1, and TCF4) resulted in short-sleeping flies, and three (MRAS, HDAC9, and TDRKH) resulted in long-sleeping flies. Short-sleeping fly lines impacted cardiac physiology consistent with links between short or poor-quality sleep and increased CVD in humans. Conversely, heart-specific disruption of five orthologs (RASD1, PHACTR1, CNNM2, MRAS, and TCF4) led to defects in cardiac physiology, with varied effects on sleep. Heart-specific knockdown lines that altered fractional shortening, a measure of cardiac contractility, also influenced long and short sleep states. These findings reveal bidirectional relationships between sleep states and cardiac performance, representing a potential feedback loop linking insomnia and CVD.

neuroscience↗

bioRxiv2024

Pleistocene sea-level fluctuation shapes archipelago-wide population structure in the Endangered Lord Howe Island cockroach Panesthia lata

Studies of biogeographic processes have often centred islands as model systems, yet questions remain about the role of Pleistocene sea-level fluctuations in shaping islands biodiversity. One novel, potentially informative model system is the Lord Howe Island Group of Australia. Despite the World Heritage status of this archipelago, almost nothing is known of the biogeographic origins, evolutionary distinctiveness or genetic diversity of the ecological communities across its 28 islands. In this study, we focused on the cockroach Panesthia lata, an ecologically specialized invertebrate with one of the broadest recorded distributions of any LHIG species. To investigate the influence of Pleistocene sea-level fluctuations on LHIG fauna, we explored the phylogeography of P. lata using single-nucleotide polymorphisms and complete mitochondrial genomes. Our analyses reveal that the lineage on the permanently isolated islet Balls Pyramid is highly divergent from the remaining populations, while those on the episodically connected Lord Howe, Roach and Blackburn Islands experienced gene flow during the last glacial period. These results offer the first evidence that Pleistocene land bridges allowed for overland migration across the archipelago. Further, although P. lata was believed to have been locally extirpated by rodents on Lord Howe Island, we discovered two surviving, relict populations. We also detected high levels of inbreeding in all populations, emphasizing the need for ongoing conservation management. Finally, the combination of shallow genetic structure and low diversity suggests that genetic rescue from another island may be a viable strategy to conserve the Lord Howe Island population of P. lata, as well as other species that have been similarly impacted by rodents.

evolutionary biology↗

bioRxiv2024

Enhanced RNA-targeting CRISPR-Cas technology in zebrafish

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity in mammalian cells and mouse models. Moreover, its efficiency could be improved in vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations to enhance the CRISPR-RfxCas13d system in zebrafish. We i) used chemically modified gRNAs to allow more penetrant loss-of-function phenotypes, ii) improved nuclear RNA-targeting, and iii) compared different computational models and determined the most accurate to predict gRNA activity in vivo. Furthermore, we demonstrated that transient CRISPR-RfxCas13d can effectively deplete endogenous mRNAs in zebrafish embryos without inducing collateral effects, except when targeting extremely abundant and ectopic RNAs. Finally, we implemented alternative RNA-targeting CRISPR-Cas systems with reduced or absent collateral activity. Altogether, these findings contribute to CRISPR-Cas technology optimization for RNA targeting in zebrafish through transient approaches and assist in the progression of in vivo applications.

developmental biology↗

bioRxiv2024

High-content microscopy and machine learning characterize a cell morphology signature of NF1 genotype in Schwann cells

Neurofibromatosis type 1 (NF1) is a multi-system, autosomal dominant genetic disorder driven by the systemic loss of the NF1 protein neurofibromin. Loss of neurofibromin in Schwann cells is particularly detrimental, as the acquisition of a second-hit (e.g., complete loss of NF1) can lead to the development of plexiform neurofibromas (pNF). pNFs are painful, disfiguring tumors with an approximately 1 in 5 chance of sarcoma transition. Selumetinib and mirdametinib are currently the only medicines approved by the U.S. Food and Drug Administration (FDA) for the treatment of pNFs. This motivates the need to develop new therapies, either derived to treat NF1 haploinsufficiency or complete loss of NF1 function. To identify new therapies, we need to understand the impact neurofibromin has on Schwann cells. Here, we aimed to characterize differences in high-content microscopy in neurofibromin-deficient Schwann cells. We applied a fluorescence microscopy assay (called Cell Painting) to an isogenic pair of Schwann cell lines (derived from ipn02.3 2{lambda}), one of wildtype genotype (NF1+/+) and one of NF1 null genotype (NF1-/-). We modified the canonical Cell Painting assay to mark four organelles/subcellular compartments: nuclei, endoplasmic reticulum, mitochondria, and F-actin. We utilized CellProfiler to perform quality control, illumination correction, segmentation, and cell morphology feature extraction. We segmented 20,680 NF1 wildtype and null cells, measured 894 significant cell morphology features representing various organelle shapes and intensity patterns, and trained a logistic regression machine learning model to predict the NF1 genotype of single Schwann cells. The machine learning model had high performance, with training and testing data yielding a balanced accuracy of 0.85 and 0.80, respectively. However, when applied to a new pair of Schwann cells, the models balanced accuracy dropped to 0.5, which is no better than random chance. This performance decline appears to result from morphology differences introduced by non-biological factors (cloning procedures, origin of parental cell line, and CRISPR procedures) of the second cell line pair. We plan to improve upon this preliminary model by refining the NF1 morphology signature using a broader panel of Schwann cell lines. Our goal is to apply this enhanced signature in large-scale drug screens of NF1-deficient cells to identify candidate therapeutic agents that specifically reverse the disease-associated morphology. Ultimately, we aim to identify agents that restore NF1 patient-derived Schwann cells to a phenotype resembling the NF1 wild-type and healthier state.

systems biology↗

bioRxiv2024

Plio-Pleistocene decline of mesic forest underpins diversification in a clade of Australian Panesthia cockroaches

The progressive aridification of the Australian continent, and coincident decline of mesic forest, has been a powerful driver of allopatric and environmental speciation in native species. The relictual mesic forests of the eastern seaboard now harbor a diverse group of endemic fauna, including the wood-feeding cockroaches of the genus Panesthia, which reached the continent via two separate invasions from Melanesia. The more recent of these colonization events gave rise to a group of five recognized species, occurring in mainland woodlands, sclerophylls and rainforests, as well as the forests and grasslands of the Lord Howe Island Group. Due to limited sampling in molecular studies and doubt regarding the standing taxonomy, there is little certainty about relationships among the species and poor understanding of the effects of ancient climatic changes upon their evolution. We undertook a comprehensive phylogenetic analysis of the clade, using complete mitogenomes and nuclear ribosomal markers from nearly all known morphospecies and populations. Our time-calibrated phylogenetic analyses reveal six unrecognized, highly divergent lineages, and suggest that these have arisen primarily through vicariance as rainforests fragmented during Plio-Pleistocene glacial cycles (2-5 million years ago). Ancestral niche reconstructions also evidence a tropical rainforest origin for the group, followed by at least three niche transitions into drier forest, including one associated with the singular colonization of the Lord Howe Island Group. Finally, we find evidence of frequent, parallel wing reduction, in potential association with the contraction of forest habitats into small refugia. Our results reiterate the far-reaching role of ancient aridification in driving speciation, niche expansion and morphological evolution in Australian fauna.

evolutionary biology↗

bioRxiv2023

Identifying novel links between cardiovascular disease and insomnia by Drosophila modeling of genes from a pleiotropic GWAS locus

Insomnia symptoms have been associated with cardiovascular disease (CVD), doubling the risk of incident CVD, but specific shared pathways remain poorly understood. Recently, genome-wide association studies (GWAS) identified genetic loci significantly associated with insomnia symptoms, including one locus (near ATP5G1, UBE2Z, SNF8, IGF2BP1, and GIP) that was previously linked with CVD in an independent GWAS. To evaluate the cell-autonomous role of genes within the 17q21 insomnia and CVD locus, we used Drosophila melanogaster models to perform tissue-specific RNAi knockdown of four conserved orthologues (ATPSynC, Lsn, Bruce, and Imp) in neurons and in the heart. To identify non-cell-autonomous mechanisms, we also assessed heart function in flies with neuronal-specific knockdown and sleep in flies with heart-specific knockdown. Neuronal and cardiac-specific RNAi knockdown of several of the genes conserved in Drosophila led to compromised sleep quality and impaired cardiac performance. Neuronal-specific knockdown of ATPSynC, Imp, and Lsn led to disruptions in sleep quantity and quality. Knockdown of ATPSynC and Lsn in the heart led to significantly reduced cardiac performance without and with cardiac dilation, respectively. Furthermore, Lsn and ATPSynC-suppressed hearts showed disruption in the actin-containing myofibrillar organization and led to a significantly shortened lifespan. Non-cell-autonomous effects were seen both from neurons to heart (Imp), and heart to neurons (ATPSynC and Lsn). Specifically, Imp neuronal knockdown led to a significantly compromised cardiac function, whereas knockdown of ATPSynC and Lsn in the heart led to compromised sleep characterized by increased sleep fragmentation, both accompanied by an increase in inflammation through Upd3, an inflammatory cytokine, in the heart or head, respectively. We also demonstrate disrupted cardiac function or sleep upon cardiac-specific or neuronal-specific overexpression of Upd3, respectively, showing a direct link between cardiac dysfunction and sleep disruption through inflammation. Our study reveals tissue-specific and cross-tissue consequences of Drosophila knockdown of multiple genes at this locus, providing novel insights into potential genetic mechanisms linking CVD and insomnia. Our study also highlights the key role of these four conserved genes in both sleep and cardiac function.

physiology↗

bioRxiv2023

Genome-wide coancestry reveals details of ancient and recent male-driven reticulation in baboons

Baboons (genus Papio) are a morphologically and behaviorally diverse clade of catarrhine monkeys that have experienced hybridization between phenotypically and genetically distinct phylogenetic species. We used high coverage whole genome sequences from 225 wild baboons representing 19 geographic localities to investigate population genomics and inter-species gene flow. Our analyses provide an expanded picture of evolutionary reticulation among species and reveal novel patterns of population structure within and among species, including differential admixture among conspecific populations. We describe the first example of a baboon population with a genetic composition that is derived from three distinct lineages. The results reveal processes, both ancient and recent, that produced the observed mismatch between phylogenetic relationships based on matrilineal, patrilineal, and biparental inheritance. We also identified several candidate genes that may contribute to species-specific phenotypes. One-Sentence SummaryGenomic data for 225 baboons reveal novel sites of inter-species gene flow and local effects due to differences in admixture.

evolutionary biology↗

bioRxiv2023

Aminobenzoic acid derivatives obstruct induced fit in the catalytic center of the ribosome

The Escherichia coli ribosome can incorporate a variety of non-O_SCPLOWLC_SCPLOW--amino acid monomers into polypeptide chains, but with poor efficiency. Although these monomers span a diverse set of compounds, there exists no high-resolution structural information regarding their positioning within the catalytic center of the ribosome, the peptidyl transferase center (PTC). Thus, details regarding the mechanism of amide bond formation and the structural basis for differences and defects in incorporation efficiency remain unknown. Within a set of three aminobenzoic acid derivatives-3-aminopyridine-4-carboxylic acid (Apy), ortho-aminobenzoic acid (oABZ), and meta-aminobenzoic acid (mABZ)-the ribosome incorporates Apy into polypeptide chains with the highest efficiency, followed by oABZ and then mABZ, a trend that does not track with the nucleophilicity of the reactive amines. Here, we report high resolution cryo-EM structures of the ribosome with these three aminobenzoic acid derivatives charged on tRNA bound in the aminoacyl-tRNA site (A site). These structures reveal how the aromatic ring of each monomer sterically blocks positioning of nucleotide U2506, thereby preventing rearrangement of nucleotide U2585 and the resulting induced fit in the PTC required for efficient amide bond formation. They also reveal disruptions to the "proton wire" responsible for facilitating formation and breakdown of the tetrahedral intermediate. Together, the cryo-EM structures reported here provide a clear rationale for differences in reactivity of aminobenzoic acid derivatives relative to O_SCPLOWLC_SCPLOW--amino acids and each other, and point to stereochemical constraints on the size and geometry of non-proteinogenic monomers that can be accepted efficiently by wild-type ribosomes.

biophysics↗

bioRxiv2022

Rapid generation of precision preclinical cancer models using regulatable in vivo base editing

Single nucleotide variants (SNVs) comprise the majority of cancer-associated genetic changes and can have diverse effects on protein function. Despite a comprehensive catalogue of SNVs across human cancers, little is known about their impact on tumor initiation and progression. To enable the functional interrogation of cancer-associated SNVs, we developed a murine system for temporal and regulatable in vivo cytosine base editing (iBE). The iBE mice show robust, doxycycline-dependent expression across a broad range of tissues with no evidence of DNA or RNA off-target effects. Transient iBE induction drives efficient creation of individual or multiple SNVs in intestinal, lung, and pancreatic organoids, while temporal iBE regulation allows controlled sequential genome editing. Moreover, in situ delivery of plasmid-based or synthetic sgRNAs to target tissues facilitates the simple and rapid generation of pre-clinical cancer models. Overall, iBE is a powerful in vivo platform to define and interrogate the genetic drivers of cancer.

cancer biology↗

bioRxiv2022

Redirecting RiPP biosynthetic enzymes to proteins and backbone-modified substrates

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are peptide-derived natural products that include the FDA-approved analgesic ziconotide1,2 as well as compounds with potent antibiotic, antiviral, and anticancer properties.3 RiPP enzymes known as cyclodehydratases and dehydrogenases represent an exceptionally well-studied enzyme class.3 These enzymes work together to catalyze intramolecular, interresidue condensation3,4 and aromatization reactions that install oxazoline/oxazole and thiazoline/thiazole heterocycles within ribosomally produced polypeptide chains. Here we show that the previously reported enzymes MicD-F and ArtGox accept backbone-modified monomers, including aramids and beta-amino acids, within leader-free polypeptides, even at positions immediately preceding or following the site of cyclization/dehydrogenation. The products are sequence-defined chemical polymers with multiple, diverse, non-alpha-amino acid subunits. We show further that MicD-F and ArtGox can install heterocyclic backbones within protein loops and linkers without disrupting the native tertiary fold. Calculations reveal the extent to which these heterocycles restrict conformational space; they also eliminate a peptide bond. Both features could improve the stability or add function to linker sequences now commonplace in emerging biotherapeutics. Moreover, as thiazoles and thiazoline heterocycles are replete in natural products,5-7 small molecule drugs,8,9 and peptide-mimetic therapeutics,10 their installation in protein-based biotherapeutics could improve or augment performance, activity, stability, and/or selectivity. This work represents a general strategy to expand the chemical diversity of the proteome beyond and in synergy with what can now be accomplished by expanding the genetic code.

biochemistry↗

bioRxiv2021

Synthetic lethal screening identifies existing drugs with selective viability effects on Neurofibromatosis type-1 model systems

Neurofibromatosis type 1 (NF1) is a genetic disorder associated with various symptoms including the formation of benign tumors along nerves. Drug treatments are currently limited. The MEK inhibitor selumetinib is used for a subset of cases but is not always effective and can cause side effects. Therefore, there is a clear need to discover new drugs to target NF1-deficient tumor cells. Using a Drosophila cell model of NF1, we performed synthetic lethal screens to identify novel drug targets. We identified 54 candidates, which were validated with variable dose analysis as a secondary screen. Five candidates could be targeted using existing drugs, with autophagy inhibitors (chloroquine (CQ) and bafilomycin A1) showing the greatest potential for selectively killing NF1-deficient Drosophila cells. When further investigating autophagy-related genes, we found that 14 out of 30 genes tested had a synthetic lethal interaction with NF1. These 14 genes are involved in the regulation of all aspects of the autophagy pathway and can be targeted with additional autophagy drugs, although none were as effective as CQ. The lethal effect of autophagy inhibitors was conserved in a panel of human NF1-deficient Schwann cell lines, highlighting their translational potential. The effect of CQ was also conserved in a Drosophila NF1 in vivo model and in a xenografted NF1-deficient tumor cell line in mice, with CQ treatment resulting in a more significant reduction in tumor growth than selumetinib treatment. Furthermore, combined treatment with CQ and selumetinib resulted in a further reduction in NF1-deficient cell viability. In conclusion, NF1-deficient cells are vulnerable to disruption of the autophagy pathway. This pathway represents a promising therapeutic target for NF1-associated tumors, and CQ was identified as a promising candidate drug for the treatment of NF1 tumors.

cancer biology↗

bioRxiv2021

Drag-and-drop genome insertion without DNA cleavage with CRISPR-directed integrases

Programmable and multiplexed genome integration of large, diverse DNA cargo independent of DNA repair remains an unsolved challenge of genome editing. Current gene integration approaches require double-strand breaks that evoke DNA damage responses and rely on repair pathways that are inactive in terminally differentiated cells. Furthermore, CRISPR-based approaches that bypass double stranded breaks, such as Prime editing, are limited to modification or insertion of short sequences. We present Programmable Addition via Site-specific Targeting Elements, or PASTE, which achieves efficient and versatile gene integration at diverse loci by directing insertion with a CRISPR-Cas9 nickase fused to both a reverse transcriptase and serine integrase. Without generating double stranded breaks, we demonstrate integration of sequences as large as [~]36 kb with rates between 10-50% at multiple genomic loci across three human cell lines, primary T cells, and quiescent non-dividing primary human hepatocytes. To further improve PASTE, we discover thousands of novel serine integrases and cognate attachment sites from metagenomes and engineer active orthologs for high-efficiency integration using PASTE. We apply PASTE to fluorescent tagging of proteins, integration of therapeutically relevant genes, and production and secretion of transgenes. Leveraging the orthogonality of serine integrases, we engineer PASTE for multiplexed gene integration, simultaneously integrating three different genes at three genomic loci. PASTE has editing efficiencies comparable to or better than those of homology directed repair or non-homologous end joining based integration, with activity in non-dividing cells and fewer detectable off-target events. For therapeutic applications, PASTE can be delivered as mRNA with synthetically modified guides to programmably direct insertion of DNA templates carried by AAV or adenoviral vectors. PASTE expands the capabilities of genome editing via drag-and-drop gene integration, offering a platform with wide applicability for research, cell engineering, and gene therapy. One Sentence SummaryA new technology combining CRISPR-mediated genome editing and site-specific integrases enables efficient programmable gene integration at any targeted genomic locus without double-strand DNA breaks, leading to broad applications in basic science research, cell engineering, and gene therapy.

bioengineering↗

bioRxiv2021

KRAS is vulnerable to reversible switch-II pocket engagement in cells

Current small molecule inhibitors of KRAS(G12C) bind irreversibly in the switch-II pocket, exploiting the strong nucleophilicity of the acquired cysteine as well as the preponderance of the GDP-bound form of this mutant. Nevertheless, many oncogenic KRAS mutants lack these two features, and it remains unknown whether targeting the switch-II pocket is a practical therapeutic approach for KRAS mutants beyond G12C. Here we use NMR spectroscopy and a novel cellular KRAS engagement assay to address this question by examining a collection of SII-P ligands from the literature and from our own laboratory. We show that the switch-II pockets of many GTP hydrolysis-deficient KRAS hotspot (G12, G13, Q61) mutants are accessible using non-covalent ligands, and that this accessibility is not necessarily coupled to the GDP state of KRAS. The results we describe here emphasize the switch-II pocket as a privileged drug binding site on KRAS and unveil new therapeutic opportunities in RAS-driven cancer.

cancer biology↗

bioRxiv2020

Prime Editing in Mice Reveals the Essentiality of a Single Base in Driving Tissue-Specific Gene Expression

Most single nucleotide variants (SNVs) occur in noncoding sequence where millions of transcription factor binding sites (TFBS) reside. Several genome editing platforms have emerged to evaluate the functionality of TFBS in animals. Here, a comparative analysis of CRISPR-mediated homology-directed repair (HDR) versus the recently reported prime editing 2 (PE2) system was carried out in mice to demonstrate the essentiality of a single TFBS, called a CArG box, in the promoter region of the Tspan2 gene. HDR-mediated substitution of three base pairs in the Tspan2 CArG box resulted in 20/37 (54%) founder mice testing positive for the correct edit. Mice homozygous for this edit showed near loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Using the same protospacer, PE2-mediated editing of a single base in the Tspan2 CArG box yielded 12/47 (26%) founder mice testing positive for the correct edit. This single base substitution resulted in [~]90% loss of Tspan2 expression in aorta and bladder with no change in heart or brain. Targeted sequencing demonstrated all PE2 and HDR founders with some frequency of on-target editing. However, whereas no spurious on-target indels were detected in any of the PE2 founders, many HDR founders showed variable levels of on-target indels. Further, off-target analysis by targeted sequencing revealed mutations in 5/11 (45%) HDR founders but none in PE2 founders. These results demonstrate high fidelity editing of a TFBS with PE2 and suggest a new paradigm for Cre/loxP-free tissue-specific gene inactivation via single base substitution in a TFBS. The PE2 platform of genome editing represents a powerful approach for modeling and correcting relevant noncoding SNVs in the mouse.

genetics↗