Jackson Laboratory

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY Aging leads to widespread changes in the immune system, including increased inflammation, diminished vaccine responsiveness, and greater susceptibility to infection and chronic disease. However, older adults vary significantly in their immune trajectories—some maintain robust function, while others experience immune decline. The biological mechanisms driving this heterogeneity remain poorly understood, particularly the role of upstream hematopoietic stem and progenitor cells (HSPCs), which continually regenerate the immune system throughout life. This project tests the central hypothesis that epigenetic remodeling of HSPCs contributes to immune-related hallmarks of aging and variability in these phenotypes, shaping both inflammatory set points and immune responsiveness in older adults. To address this, we leverage a well-characterized, longitudinal cohort of 62 older adults, each of whom received a different influenza vaccine over three consecutive seasons. Using our novel PBMC-PIE platform—developed to enrich and profile circulating HSPCs from cryopreserved PBMCs at single-cell resolution—we can interrogate progenitor cell states and their progeny without requiring bone marrow biopsies. This enables unprecedented, longitudinal insight into how human HSPCs are reprogrammed with age and vaccination. In Aim 1, we will identify molecular and epigenomic features in HSPCs and innate immune cells that distinguish strong from poor vaccine responders. In Aim 2, we will determine how different influenza vaccine platforms (high-dose, adjuvanted, recombinant) shape the chromatin landscape and lineage bias of HSPCs and their progeny over time. In Aim 3, we will test whether maladaptive “aged” HSPC states can be rejuvenated in vitro using candidate immunomodulatory factors identified in Aims 1–2. By linking blood-based epigenetic signatures to real-world immune outcomes, this work will reveal fundamental mechanisms of immune aging, identify biomarkers of immune resilience, and inform the development of targeted strategies to restore immune function in older adults.

NIH Research Projects · FY 2026 · 2026-06

PROJECT SUMMARY The research proposal addresses the urgent need for predictive biomarkers and pharmacological treatments for cocaine use disorder (CUD), a highly heritable disease affecting millions worldwide. Despite advances in understanding the genetic, cellular, and circuit mechanisms of CUD, effective treatments remain elusive. The proposal highlights the gut microbiome as a significant behavioral modifier, influencing brain function and behavior through metabolites like short-chain fatty acids (SCFAs). Recent studies suggest that the gut microbiome can impact addiction-related behaviors, making it a promising target for novel therapeutic approaches. The study proposes a paradigm shift from traditional brain-centric addiction research to a focus on the gut-brain axis. Advanced techniques such as metagenomic whole genome shotgun sequencing (mWGS) and untargeted metabolomics will be used to gain functional insights into the microbiome. The approach involves using genetically diverse mouse populations to model addiction-related behaviors and study the gut-brain axis. Behavioral assays will assess novelty-seeking, anxiety, and cocaine sensitization, while microbiome and metabolomics analyses will identify genetic loci associated with these behaviors. The proposal emphasizes the integration of genetic, microbial, and neural data to construct multi-OMICS causal networks, advancing beyond associations to establish causation. Experimental validation will involve gene editing, microbiome or metabolite interventions, and fecal transplants to confirm findings and assess their translational potential. The study aims to identify specific microbes and metabolites that can modulate addiction- related behaviors, ultimately developing predictive biomarkers and therapeutic approaches for CUD. Expected outcomes include mapping quantitative trait loci (QTLs) for microbial abundance and behaviors, constructing causal networks, and identifying behavioral modifiers. The research will leverage extensive pre- existing datasets and generate new data to uncover novel molecular mechanisms in addiction. The findings will be made publicly available, facilitating further research and potential clinical applications. This comprehensive approach aims to significantly advance the field of addiction research and contribute to the development of effective treatments for CUD.

NIH Research Projects · FY 2026 · 2026-06

The proposed animal studies are essential to evaluate the in vivo effects of AML engraftment, disease progression, and therapeutic response, where systemic treatment exposure and leukemia interactions with the hematopoietic microenvironment cannot be adequately modeled in vitro. This includes the ability to assess the in vivo delivery efficiency and therapeutic potential of CpG-conjugated ASOs targeting of the long ZBTB7A 3′UTR isoform in AML. In addition, the NSG-SGM3 mice are particularly well suited for these studies because they are the most robust model to support engraftment of primary AML patient-derived xenografts, enabling faithful assessment of leukemia growth, treatment response, and survival while preserving the biologic heterogeneity of patient samples. These models are therefore required to determine the effects of ELAVL1/ELAVL4 perturbation in Aim 1 and CpG-conjugated ASO targeting of the long ZBTB7A 3′UTR isoform on leukemia burden and survival in Aim 2.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY/ABSTRACT A lack of proven interventions to prevent leukemia in aged populations leaves a growing demographic vulnerable to this devastating disease. While leukemic cells are subject to immune selection which influences disease progression, we lack knowledge of the stages at which T cells shape the hematopoietic stem and progenitor cell (HSPC) pool from the initiation of clonal hematopoiesis (CH) through to the progression to leukemia, which limits our ability to intervene in this process. The long-term goal of this project is to identify immuno-preventative strategies to intercept leukemogenesis at its earliest stages. The overall objective of this application is to determine the mechanisms by which, and at which stages of pre- leukemic development, HSPC clones are detected and selected by the adaptive immune system. The central hypothesis is that reduced IFNγ responsiveness enables immune evasion of CH-mutant (Dnmt3amut) HSPCs thereby promoting clonal expansion and pre-leukemic evolution. The rationale is grounded in the observation that HSPCs from humans and mice with recurrent CH driver mutations in Dnmt3a have reduced transcript and protein expression of MHC-II machinery, and reduced presentation of exogenous and endogenous antigens via MHC-II. Mechanistically, MHC-II is potently induced by IFNγ on wild-type HSPCs but to a lesser extent on Dnmt3amut HSPCs. In vitro and in vivo, we observe less activation and proliferation of CD4+ T cells by Dnmt3amut HSPCs compared to control HSPCs, supporting that Dnmt3amut HSPCs have reduced immunogenicity. The central hypothesis will be tested by pursuing two specific aims: 1) to define the stages of pre-leukemic HSPC selection that are controlled by CD4+ T cells, and 2) to evaluate decreased IFNγ response of pre-leukemic Dnmt3amut HSPCs as a mechanism of immune evasion. This research is innovative because it introduces a novel framework for understanding how adaptive immunity shapes clonal evolution of pre-leukemic HSPCs. While considerable attention has been given to genetic and cell-intrinsic drivers of CH, the role of immune surveillance—particularly adaptive immune selection—in governing HSPC clonality remains largely unexplored. Ultimately, the proposed work is significant because it will define the role of CD4⁺ T cells in HSPC clone selection during early disease phases in CH and pre-leukemia which has major therapeutic implications for immunoprevention of leukemia.

NIH Research Projects · FY 2026 · 2026-05

PROJECT SUMMARY Retinal vascular diseases including diabetic retinopathy (DR) are a leading cause of vision loss worldwide. Current DR treatments center on reducing retinal vascular dysfunction and neovascularization. These treatments do not improve all outcomes and can reduce visual acuity underscoring the need for novel therapeutics targeting additional aspects of DR. Early neural deficits including retinal ganglion cell loss (RGC) have been identified in both patients and in DR mouse models. Microglia have been implicated in retinal neurodegenerative conditions including DR. Recent work indicates microglial mitochondrial function and metabolic flexibility drives dysfunctional responses. Yet, while human DR is linked to metabolic and mitochondrial stress how this perturbs microglia and if this drives RGC loss and microvascular damage is not clear. The goal of this project is to identify and test mechanisms by which microglia influence RGC and retinal vascular health in a DR-relevant context. Our recent work identified NZO mice (a polygenic model of metabolic syndrome) as a novel model of DR-relevant metabolic syndrome associated retinopathy. NZO mice develop hallmarks of human DR including cotton wool spots, exudates, and capillary loss. In particular, NZO mice develop microvascular dysfunction concomitant with neural deficits (initiates by 3mo) and significant RGC dysfunction and loss by 12mo. Preliminary data from single nucleus and single cell RNA-seq experiments comparing NZO (retinopathy) and B6J mice (control) suggested microglia differentially influence RGCs in B6J compared to NZO retinas. Further, NZO microglia exhibited age- dependent changes in diabetic wound healing genes, and lower expression of metabolic genes compared to B6J microglia suggesting metabolic dysregulation may underlie abnormal microglia-RGC communication. Therefore, in Aim 1, I will identify mechanisms by which microglia influence RGCs and the vascular cells using single cell RNA-seq, microglia depletion, and data alignment strategies. Additional preliminary analyses of our mouse datasets and available patient data indicate lower expression of nicotinamide metabolic enzymes in RGCs and microglia reinforcing the possibility that metabolic dysfunction may contribute to retinopathy- progression. Thus, in Aim 2 I will examine the therapeutic potential of improving cellular metabolism in NZO mice through nicotinamide and pyruvate supplementation or by increasing expression of nicotinamide mononucleotide adenylyl transferases in RGCs and microglia via AAVs. In Aim 3, in a candidate target approach I will first determine the impact of advanced glycation end products signaling in microglia on DR progression. I will then take an unbiased candidate prioritization approach and utilize data generated in Aim 1 to identify and test additional human-relevant processes that may drive pathology. Through these Aims, I will acquire essential skills to facilitate my transition to an independent investigator studying the role of microglia in retinal disease. This project will be supported by a mentoring committee of globally recognized experts in retinal neurodegeneration, microglia, and bioinformatics and excellent career development resources.

NSF Awards · FY 2026 · 2026-05

This REU Site award to The Jackson Laboratory (JAX), located in Bar Harbor, ME, will support the training of 10 undergraduate students for 10 weeks during each summer in 2026-2028. It is anticipated that a total of 30 students from institutions across the United States will be trained in the program to conduct hypothesis-driven research in genetics and functional genomics with JAX research mentors who lead active and collaborative research programs. The program supports student development of highly sought-after computational and data analysis skills. Students will learn how research is conducted, and many will present the results of their work at scientific conferences. Program assessment includes the common BIO REU survey questions and questions adapted from the SURE survey. Students should apply to the REU site using NSF ETAP (Education and Training Application: https://etap.nsf.gov). Students will be tracked after the program in order to determine their career paths. The training students will receive is aligned with the NSF priorities in AI and Biotechnology. The REU Site curriculum includes: an online course and certificate program through JAX-developed Minicourses, in-person coding workshops, a formal journal club, professional development seminars, and workshops on the ethical, legal, and social issues in genetics research including workshops around communicating science to different audiences. The REU cohort will join the JAX Summer Student Program, a Living Learning Community for up to 30 students per year with a well-established structure. Possible student projects are in the areas of computational strategies using genetic data to understand complex genetic systems, fundamental questions in genetics and evolution, how protein glycosylation influences cellular signaling, using modern computer vision tools to connect genetics to tissue structure and organ function, and the genetics of complex traits. Applications are reviewed by a team of professional JAX scientists on the basis of the candidate’s research motivation as well as the candidate’s ability to work independently and yet collaboratively. Researchers inviting students into their labs for the summer make the final student selections. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Dementias are projected to become the most burdening group of diseases, expected to affect over 150 million people worldwide by 2050, and Alzheimer’s disease (AD) is the most common among them. Currently, over 6 million Americans are living with AD. While there have been several recently-approved drugs for AD, these drugs target amyloid beta plaques – just one facet of the disease – and have not proven effective in all patients. Non-neuronal cell types in the brain such as microglia (the brain’s immune cells) and astrocytes (star- shaped helper cells) play a critical role in disease progression but have been traditionally understudied. Microglia are known to be activated by amyloid beta plaques and they were ascribed both a protective and a detrimental role. They were shown to induce a toxic state in astrocytes. The microglia’s behavior is likely influenced by patient genetics, which might explain why the majority of AD risk genes are expressed in microglia. To determine which of the 90 known AD-associated genetic variants exert their effect through microglia, we need a better understanding of the functional links between a variant and the disease. This requires large-scale studies of diseased, human cells from genetically diverse patients, as there is a wide variety of genetic variants that can influence AD risk. This project proposes to develop an automatable protocol for the creation of human induced pluripotent stem cell (iPSC)-derived microglia (iMG) and their co-culture with primary astrocytes. Standardized co-cultures of iMGs from AD patients and primary astrocytes will allow scientists to observe how these cells interact in a diseased environment, better understand known variants, and possibly identify new risk variants. Attaining sufficient statistical power for the identification of novel variants requires many cell lines, which can only be achieved with robotic automation. This study will serve as a proof of concept to demonstrate that such co-culture systems be automated and will later be scaled up to include additional cell lines. It will furthermore help to understand how certain genetic variants contribute to AD, which might inspire new therapeutic approaches.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY The objective of this proposal is to elucidate the complex genetic, transcriptional, and histological landscape underpinning palate morphogenesis by leveraging a systems genomics approach in genetically diverse mice. Craniofacial morphogenesis involves the outgrowth and fusion of facial prominences, which must be coordinated with skeletal specification and differentiation to generate a functional craniofacial complex. Underscoring the sensitivity of this intricate choreography, orofacial clefts (OFCs) are the most common facial anomaly in humans, affecting 1/700 births worldwide. Mouse models provide a key platform for human disease allele discovery owing to recent developments in gene editing that enable rapid validation of novel variants. However, multiple published examples demonstrate the profound effect that mouse genetic background can have on the penetrance and expressivity of craniofacial phenotypes. Thus, our limited understanding of the effect of natural strain variation on developmental processes presents a major challenge to validation of disease variants and our ability to model human congenital malformations. While individual genes are critically required for normal development, it is the collective function of genes and their interactions, modularly organized into gene regulatory networks (GRNs), that control the transcriptional dynamics and timing of morphogenesis and differentiation. Variation in these dynamics likely accounts for normal variation in facial shape but can also underpin craniofacial birth defects. However, feasibility limits studies of genetic variation in developing embryos to only a few genes at a time and experimental models that accurately relate genomic sequence variation to variation in transcriptional dynamics are critically lacking. These challenges complicate a systematic address of genome-scale mechanisms that drive morphogenesis and the potential for pathological outcomes. The complementary Collaborative Cross (CC) recombinant inbred strains and Diversity Outbred (DO) heterogeneous stock combine the genetic and phenotypic diversity of 8 common inbred founder strains of mice in a design optimized for systems-level genetic dissection of complex traits. It has been demonstrated that QTLs for facial shape in adult DO mice are enriched for genes with established function in craniofacial and skeletal development. However, these studies do not address mechanisms of where, when, or how QTL candidate genes influence facial shape. The proposed aims will apply a developmental systems genomics approach to map the influence of genetic variation within a model of palate morphogenesis. Aim 1 will define the temporal dynamics of morphogenetic networks within the segmentally organized upper jaw and identify QTL/eQTL underlying strain differences in developmental timing and palate morphogenesis. Aim 2 will employ single-cell and spatial genomics to genetically dissect the molecular signatures of QTL/eQTL with cellular and histological resolution. Aim 3 will leverage quantitative phenotyping to relate differences in morphogenetic dynamics and resulting morphology to variation in underlying genotype and thereby derive mechanistic insight into the integration of genomic regulatory systems.

NIH Research Projects · FY 2026 · 2026-03

Spatial transcriptomics (ST) is a rapidly evolving field with great potential to shape our understanding of cells as actors in human health and disease by integrating cellular expression with position to illuminate the spatial organization of gene expression and inter-cellular communication. While ST technologies hold great promise for researchers, the complexity of the analytical methods creates a high entry barrier into the field, leaving researchers unable to take advantage of ST’s explanatory power. Some barriers to researchers seeking to adopt these technologies include insufficient access to in-person courses, lack of support in online tutorials, or a focus on less widely used technologies or programming languages. To train researchers to apply ST methods, we propose The Short Course on Spatial Transcriptomics at The Jackson Laboratory (JAX). This four-day course will focus on the fundamentals of ST technologies, applications, experimental design, and analysis of 10X Genomics Visium data in the R programming language, the most widely used technology and programming language in ST publications. The course will target researchers in neuroscience, cancer, development, genetics, and biomedical research at academic levels ranging broadly from graduate students to established researchers. Our goal is to build an educational program that recruits and cultivates new expertise, teaches rigorous, reproducible practices, and creates a nurturing climate and supportive network that builds a community while lowering barriers to entry into this rapidly emerging field. Achieving our goal will advance cutting-edge research at the Forefront of Genomics. While furthering NHGRI priorities, the proposed course will also promote rigorous, reproducible ST practices, create a collaborative network of ST practitioners, and deliver critical skills and practice for collaborative team training.

NIH Research Projects · FY 2026 · 2026-03

PROJECT SUMMARY Post-transcriptional regulation is a critical mechanism controlling cellular differentiation and development, driven by coordinated interactions between RNA-binding proteins (RBPs) and RNA modifications. This proposal investigates the interplay between Quaking (QKI) isoforms and N7-methylguanosine (m7G) RNA modifications. QKI isoforms exhibit distinct subcellular localizations and functions, yet their roles in binding m7G-modified transcripts at 3′ untranslated regions (UTRs) during differentiation remain poorly understood. The central hypothesis is that QKI isoforms regulate differentiation by binding m7G-modified mRNAs at 3′ UTRs, with cytoplasmic QKI6 stabilizing transcripts essential for myeloid differentiation. Furthermore, m7G modifications may independently regulate gene expression in ways yet to be defined. Our preliminary evidence demonstrates that cytoplasmic QKI isoforms mediate myeloid differentiation, highlighting a critical gap in understanding m7G’s functional roles in steady-state cellular processes. Current methods for detecting m7G methylation at single- nucleotide resolution face technical limitations. This project addresses these challenges by employing direct RNA long-read sequencing and orthogonal methods to map m7G modifications with precision and identify QKI isoform-specific mRNA targets. Integrating these approaches will elucidate how QKI-m7G interactions influence myeloid differentiation, with broader implications for RNA modifications in stem cell biology, neurodevelopment, and cancer. Over the next five years, the laboratory’s mission is to (1) develop novel methods for base-resolution m7G detection, (2) define mRNA targets regulated by distinct QKI isoforms, and (3) determine the functional impact of QKI-m7G interactions on myeloid differentiation. These studies will advance understanding of RNA modification-driven gene regulation and may inform therapeutic strategies for diseases such as leukemia. By resolving the interplay between QKI and m7G at 3′UTRs, this work will reveal how their coordination regulates cellular functions across human cell types, directly addressing NIGMS’s mission to support foundational discovery science. The proposed research will provide mechanistic insights into RNA modifications dysregulated in cancer, potentially uncovering new targets for therapeutic intervention in malignancies and developmental disorders.

NIH Research Projects · FY 2026 · 2025-12

PROJECT SUMMARY Microglia are the macrophages of the brain and become activated in response to amyloid. Recently, single cell sequencing has defined multiple activated states of microglia including two states that are robustly induced in animals of Alzheimer’s disease (AD): disease associated microglia (DAM), and interferon responding microglia (IRM). It is now established that in response to amyloid microglia initiate the classical pathway of the complement cascade, and that the complement cascade is a critical mediator of neuronal synapse loss during disease progression. Synapse loss is among the strongest neurobiological correlates of cognitive decline in AD. Global ablation of the C1 complex (via C1qa gene knockout) preserves synapses in AD mouse models, highlighting the importance of determining the mechanisms determining the role of microglia in complement-mediated synapse loss. Yet despite much work, key knowledge gaps remain. First, the relationships among the different transcriptionally defined microglia states have not been determined. Second, all microglia express C1Q and it remains unknown whether microglia belonging to distinct states trigger synapse loss on neurons. Third, the complement cascade requires downstream components such as complement factors C2 through C9 that are not expressed by microglia, but virtually nothing is known about the spatial and temporal coordination of the specific cell types expressing these components in the brain. Filling these knowledge gaps may lead to new therapeutic avenues that prevent or intervene in synapse loss in AD. By leveraging floxed alleles of Csf1r, Trem2, Sting1, C1qa, C3, C5 and C7, microglia state specific Cre driver lines such as Cx3cr1-cre, Tmem119-cre, Itgax-cre, and Mx1-cre, and reporter lines to lineage trace distinct states, we will take a multi-modal approach based on genetic strategies to address these questions with cellular specificity. We will use distinct mouse genetic contexts we have shown are susceptible (C57BL/6J) or resilient (PWK/PhJ) to synapse loss, and we will employ state-of-the- art methodologies including single cell myeloid cell sequencing, spatial transcriptomics and protein visualization, and circuit-specific labeling of synapses. In three aims we will test the model that IRM are an intermediate microglia state necessary to recruit DAM to plaques, and that DAM are the critical state driving complement- mediated synapse loss. In Aim 1, to test whether IRM are the intermediate state between homeostatic microglia and DAM, we will lineage trace IRM, ablate DAM or IRM, and conditionally delete Sting1 (a key mediator of interferon signaling) from DAM. In Aim 2, to determine whether DAM are the primary initiators of complement mediated synapse loss, we will conditionally delete Trem2 from homeostatic microglia, ablate DAM, and conditionally delete C1qa from DAM. In Aim 3, to uncover the cell types producing the downstream components of the complement cascade, we will perform spatial transcriptomics and protein visualization. We will then conditionally delete a downstream component from its parent cell type. Successful completion of these aims will result in the identification of critical cellular and genetic contributors to complement-mediate synapse loss in AD.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY Cell polarity mechanisms are essential for shaping the morphology and orientation of hair cells in the inner ear, ensuring proper sensory function. Communication at apical junctions between hair cells and their glial neighbors, the supporting cells, is particularly important for proper hair cell orientation. While supporting cells are known to relay polarity signals between hair cells, the possible influence of polarity mechanisms on their own differentiation is not well-studied. Pillar and Deiter's cells, specific types of supporting cells, have a complex and asymmetrical morphology beneath the epithelial surface. These cells develop apical extensions called phalangeal processes, which interdigitate between hair cells and play a critical scaffolding role in cochlear amplification and thus auditory function. The proposed research hypothesizes that mouse Pillar and Deiter's cells acquire their phalangeal processes through a postnatal polarization program similar to the polarization of hair bundles in hair cells. Preliminary results suggest that the basal body of supporting cells is planar-polarized and can exhibit a highly dynamic behavior during development. We aim to track the position of the basal body in supporting cells from embryogenesis to adulthood, correlate its position with the emergence of dense microtubule arrays in the underlying phalangeal process, and investigate defects in one relevant cell polarity mutant context. Additionally, we aim to develop methods for sparse labeling of supporting cells to track their individual morphology and analyze structural changes during phalangeal process development. Examining the polarization of supporting cells offers a novel perspective to address their complex remodeling, a neglected aspect of auditory science where new approaches and tools have potential for significant discoveries. Beyond providing a better fundamental understanding of supporting cell development, this groundwork will enable the analysis of morphometric changes in mutants causing congenital hearing loss. This work also has broader potential for studying supporting cell integrity in various conditions including aging and trauma such as noise damage.

NIH Research Projects · FY 2025 · 2025-09

This proposal seeks support for a symposium, Immuno-Cardiology 2025. Immunology is of great importance to cardiovascular research since inflammation and fibrosis are indicators of both heart failure progression and myocardial remodeling, topics at the core of NHLBI’s research mission. The involvement of the immune system in health and disease is currently an area of intense interest in the field, requiring innovative, interdisciplinary research to realize the potential for transforming the landscape of cardiovascular medicine. Cardiovascular disease is the leading cause of death in the United States, and improving our understanding of the role of inflammation in heart disease will improve patient outcomes. We hosted a highly successful inaugural conference on the topic of Immuno-cardiology in September 2023 that featured recent advances in the basic science and clinical relevance of dysregulated immune activation and fibrosis in response to cardiac injury, presented by an unprecedented cross-section of established and emerging leaders in their areas of expertise. The conference attracted a capacity audience of over 100 participants. Our long-term goal is to facilitate collaborations and growth in this important field. Specific Aims of the symposium: 1) Build on the momentum of the 2023 Symposium, bringing together various expertise in the field to continue exploration of synergies and opportunities for cross-fertilization and translation in this fast-moving field; 2) Integrate findings from related organ systems (kidney, lung and liver); 3) Foster relationships to encourage new collaborations; 4) Attract interest and participation from the next generation of scientists who will transform this interdisciplinary field. The meeting is unique in that it will bring together academic, clinical and pharmaceutical-based scientists, include researchers looking at relevant organ systems other than the heart, and showcase the work of young investigators. NIH support for the symposium will specifically provide travel funding to promote participation by early career scientists, graduate students and postdoctoral fellows.

NIH Research Projects · FY 2025 · 2025-09

PROJECT SUMMARY / ABSTRACT The goal of this proposal is to establish whether age-related functional changes in peripheral immune cells influence Alzheimer's disease (AD) pathology onset and progression. While immune dysfunction is a hallmark of AD pathogenesis, peripheral-central nervous system crosstalk is not completely understood, creating a barrier to the development of new therapeutics for prevention or cure. The foundation of proper immune responses is the hematopoietic system's ability to produce well-functioning cells in correct proportions. Hematopoietic stem cells (HSCs), which produce all blood cells (including monocytes, neutrophils, T cells, and B cells), accumulate somatic mutations over their long lifetimes. Certain mutations provide a selective advantage that results in an increased expansion of those HSC “clones” and can lead to skewed proportions of and phenotypic changes in produced cell types. The incidence of this phenomenon, termed clonal hematopoiesis (CH), increases with age and is associated with higher risk of multiple diseases. Surprisingly, CH has been recently reported to be associated with decreased incidence of AD. To validate this association and propose an actionable mechanism for clinical disease intervention, we need to understand how mutations in peripheral immune cells impact the etiology and progression of AD. These studies necessitate mammalian models in which CH and AD proteinopathy are inducible in the same individual and blood and brain are available for examination prior to disease onset and throughout disease progression. Our research objective is to investigate the impact of Tet2 CH mutation on the ability of peripheral immune cells to infiltrate the brain and modulate AD pathology. To achieve our aims, we will introduce mutated cells into the circulation of well-defined AD mouse models. We will assess the effect of aging and proteinopathy development on the extent CH peripheral cell infiltration, identify and quantify infiltrating immune cells and their locations within the brain, determine whether circulating CH cells are increasingly primed for mobilization, and assess the effect of CH on AD pathology development. Determining that a CH mutation a) increases the immune cell infiltration into the brain, and b) leads to deceased pathology would provide a direct link between CH and AD and may nominate new candidates for therapeutic intervention for AD.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY/ABSTRACT Iron disorders are caused by changes in iron levels in blood, tissues, or both. Iron deficiency, which can lead to anemia, represents a major component of the global disease burden worldwide, especially in women. Iron deficiency-related anemia affects >1.2 billion individuals worldwide, and iron deficiency without anemia is even more frequent. Understanding the basic molecular mechanisms underlying specific iron disorders is critical to designing future therapeutic strategies. Approaches using iron supplementation or iron chelators are employed globally to improve iron metabolism. However, their effectiveness can vary among patients due to the diverse mechanisms underlying iron disorders. Some etiologies are linked to impaired iron absorption in the intestine, while others involve issues with iron delivery to the bone marrow, which is crucial for sustaining erythropoiesis. The goal of my program at The Jackson Laboratory is to identify the root causes of iron disorders, including iron deficiency, iron deficiency anemia, anemia, and/or iron overload, by holistically investigating the fundamental biological mechanisms of iron metabolism. By focusing on the etiology of these disorders rather than merely addressing the symptoms, we aim to facilitate personalized medicine tailored to each patient’s specific needs. During the next five years, the AgoroLab will develop a novel computational framework, the Multi-Omics-Multi- Organ sequencing (MOMO-seq), for studying mammalian iron systems biology in different tissues associated with iron metabolism phenotypes and identify key organs, cells, and molecular features that drive iron disorders. MOMO-seq couples the power of single-cell multi-omics technology to profile DNA accessibility with transcriptomics/histopathology approaches to study iron biology across multiorgan systems, enabling the identification of cell-specific mechanisms and inter-organ communication circuits associated with iron metabolism. Within our framework, we will be able to shed light on some of the key yet unanswered questions in iron biology. This includes deconvoluting the complex multiorgan signaling network that maintains iron homeostasis. For instance, how does the bone marrow communicate with the spleen to meet the high iron demand of erythropoiesis? How do kidneys regulate iron reabsorption? Developing MOMO-seq will be pivotal in establishing and strengthening my independent research program, as well as laying the groundwork for an innovative, comprehensive approach to studying iron metabolism and its implications in health and disease.

NIH Research Projects · FY 2025 · 2025-08

PROJECT SUMMARY Re-training supplement request for R37NS054154 under NOT-OD-23-170 The overall goal of R37NS054154 is to understand the molecular mechanisms through which dominant mutations in tRNA synthetase genes cause Charcot-Marie-Tooth type 2D and related inherited peripheral neuropathies. Towards this, we will 1) examine the biochemical interactions of the mutant synthetases and their cognate tRNAs, 2) explore why only alpha motor neurons and a subset of sensory neurons are affected by these mutations, and 3) test the therapeutic potential of inhibiting the integrated stress response, which is activated by these mutations. Much of this work has been done in mouse models of these diseases, but to extend these studies into a human system, we have engineered GARS/CMT2D mutations into a healthy control iPSC line, KOLF2, which we can differentiate into motor neurons in vitro. We have encountered two challenges with this experimental system. First, the motor neurons are still immature in culture. We have assessed this in part by testing the extent to which they turn on eEF1A2 and turn off eEF1A1. This developmental switch occurs in the first few weeks of life in mice, correlating with the onset of neuropathy in our Gars mice, and we believe it to be central to the cell-type specificity of tRNA synthetase mutations, since eEF1As directly interact with tRNA synthetases and charged tRNAs. Thus, we need to develop sophisticated hiPSC co-culture systems to generate more mature motor neurons that switch more completely to eEF1A2. Second, inhibiting the integrated stress response in our mouse models is highly efficacious, but the rapid response to inhibition and neurophysiological improvements suggest this benefit is coming from improved transmission at neuromuscular junctions. However, whether NMJs are perturbed in the human disease is unknown. The best model for testing this before moving to patients is an hiPSC-derived nerve-muscle co-culture system where we can establish that motor neuron- muscle connectivity is indeed perturbed and that treatment with integrated stress response inhibitors improves function. Establishing this would make ISR inhibitors more translational and increase interest in CMT from companies developing such drugs. These more sophisticate co-culture systems and particularly those for evaluating NMJ connectivity, are beyond the current expertise of the Burgess lab. We therefore propose this re- training supplement request to train Dr. Timothy Hines in these approaches. Dr. Hines is an accomplished senior postdoc who will transition to an Associate Research Scientist position at JAX to do this work. We have assembled a team of local and external mentors to train Dr. Hines in these methods. With these skills, Dr. Hines will be well-positioned to find an independent position and secure NIH funding in the future. This supplement will benefit Dr. Hines’ professional development and the parent grant by making the experiments more translationally relevant.

NIH Research Projects · FY 2026 · 2025-05

PROJECT SUMMARY / ABSTRACT Stroke causes long-term deficits in motor function, making it the leading cause of adult disability in the US. The brain exhibits endogenous plasticity, yet these responses are insufficient in restoring full function. Recently, we have shown that motor recovery involves recruitment of transcriptional programs for memory and targeting these programs can significantly boost recovery. However, a significant gap remains in our understanding of how molecular programs in learning can alter circuit function after stroke. Execution of motor function requires interactions across various cortical and sub-cortical structures. The studies proposed will close a significant gap in our understanding on how motor recovery that requires cross-regional circuit dynamics can be reinstated by drawing from circuit mechanisms underlying learning and memory, where plasticity mechanisms operant in learning can facilitate motor recovery. Using a combination of large-scale two photon calcium imaging, high- resolution behavioral phenotyping, viral approaches to tag ensembles and transcriptomics, studies will uncover a novel circuit mechanism post-stroke that can be targeted for repair. Studies will test if, functional allocation- a process by which highly excitable neurons at the time of a new incoming stimulus are allocated with storing information for that stimulus, can be applied to the stroke brain to restore function. To determine if functionally allocated neurons are required for post-stroke recovery, studies in Aim 1 will functionally tag neurons that are active during reaching, perturb the same neurons optogenetically, and transcriptionally profile these neurons and other cell-types. Aim 2 will investigate how molecular perturbation of plasticity pathways impacts spatiotemporal and longitudinal activity changes in functionally allocated neurons and how these drive local connectivity. Finally in Aim 3, we will utilize large-scale neural activity recordings across multiple cortical regions to determine how functional allocation post-stroke modulates mesoscale cortical dynamics in skilled motor function. Together, the studies proposed will uncover a novel circuit mechanism for repair that can be modulated with existing drugs to target post-stroke motor function.

NIH Research Projects · FY 2026 · 2025-04

PROJECT SUMMARY Inbred mouse strains are integral tools for both preclinical and basic research. While the absence of genetic diversity is a widely perceived strength of inbred strains, it is also a critical limitation, as a single inbred genetic background cannot capture the full spectrum of phenotypic responses observed in genetically diverse human populations. This limits the translation success from inbred mouse models to human clinical outcomes and underscores the need for mouse platforms that more accurately model human genetic variation. The Diversity Outbred (DO) mouse population was founded in 2012 to meet these crucial research needs. The DO was initiated from eight genetically diverse inbred mouse strains, including wild-derived representatives from each of three cardinal house mouse subspecies, and has been maintained by pseudo-random mating for over 50 generations. However, the inclusion of founder strains derived from reproductively isolated subspecies has, over time, exposed genetic incompatibilities and unchecked selfish elements, resulting in allele frequency distortions that have compromised the genetic integrity of the DO. Here, we propose to generate a new, high diversity outbred mouse population, the Wild Mouse Diversity Panel (WMDP), founded from novel wild-derived inbred strains of single subspecies origin (Mus musculus domesticus). Our recent work has shown that a large proportion of variants in the genomes of these newly developed M. m. domesticus strains are absent from existing inbred mouse strains, and that these strains capture novel neurobehavioral, metabolic, physiological, and biochemical phenotypes not observed in common laboratory mouse models. Thus, the WMDP stands to minimize potential haplotype distortion due to genetic incompatibilities between subspecies, allow functional assessment of millions of variants that have never been tested in the laboratory, and empower investigations of natural genome complexity. In Aim 1, we will initiate an outbred population from four phenotypically and genetically diverse wild- derived M. m. domesticus inbred strains, implementing routine genomic monitoring of allele frequencies to ensure the genetic integrity and longevity of the WMDP resource. To aid discovery efforts in this new population, we will generate key genomic resources for the founder strains in Aim 2, including high quality de novo genome assemblies, comprehensive variant call sets, and a gene expression atlas of several tissues. In Aim 3, we will provide a proof-of-principle study to underscore the power and utility of the WMDP. We will profile phenotypic diversity for several morphological, metabolic, behavioral, and clinical traits in early outbreeding generations of the WMDP, map causal loci, and integrate our findings with human data to identify human-mouse translational parallels at the levels of both genes and gene networks. This project will yield a new outbred mouse population poised for near limitless discovery in all areas of basic and preclinical research. This resource will become part of the Special Mouse Strain Resource at The Jackson Laboratory, which will provide the framework and setting for its long-term maintenance and distribution to the global research community.

NIH Research Projects · FY 2025 · 2025-01

PROJECT SUMMARY / ABSTRACT Large-scale molecular datasets have great potential to yield new biological discoveries and accelerate biomedical research. However, specialized skills and training are needed to effectively access, integrate, and interpret them. This project will address a shortage of these skills, by providing training and exposing students to examples of successful reuse of large-scale data. To equip researchers with the necessary technical, scientific, and implementation-related skills, we propose the Short Course on Methods for MultiOmics Data Analysis at the Jackson Laboratory (JAX). This week-long course will focus on developing conceptual knowledge and practical analytical techniques centered around rigorous analysis of Common Fund datasets. Our course will also focus on building the soft skills needed to work effectively in collaborative research teams. Experts from Common Fund projects including SenNet, KOMP2, GTEx, and the Metabolomics Workbench will serve as instructors and mentors in the course. Our goal is to build an educational program that cultivates rigorous, reproducible practices in managing and analyzing large-scale molecular data from the Common Fund while fostering effective, productive research teams and long-term collaborations. Achieving these goals will strengthen scientific rigor and broaden the impact of Common Fund data sets.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY Abrogating immune checkpoint control has proven to be a powerful therapeutic strategy against cancer with documented long-term remissions in refractory metastatic disease. However, only a subset of patients (10-40%) have meaningful responses. While intrinsic tumor factors play a role in predicting immune checkpoint inhibitor (ICI) outcomes, germline variations in host immune genes are also likely to have a significant impact given their importance in defining immune response and autoimmunity. That both the tumor and the host genomes vary makes the identification of germline factors modifying ICI response highly challenging. We have solved this problem by devising a mouse experimental platform that “fixes” the genomic configuration of the tumor as a transplantable cell line (MC38) while varying germline host genetics, thus permitting the unbiased mapping of quantitative trait loci (QTL) for anti-tumor response after anti-PD1 (aPD1) therapeutics. By crossing Collaborative Cross (CC) multiparent recombinant inbred lines with the C57BL/6 strain, the resultant F1s have genetic variability but will accept the MC38 transplant. In preliminary data, we show that host genetics accounts for 42% of the variation in aPD1 response (heritability, H2) and have mapped aPD1 response to four QTLs on mouse (m)Chr 5, 9, 15, and 17 enriched for immune genes. By selective intercrosses between responder and non- responder CC lines, we found significant epistatic interactions between chromosomes 5 and 17 and between 15 and 17. We devised a cross-species tumor microenvironment (CST) algorithm that reduced the ~1,500 genes in the QTLs to 48 top candidate genes that could predict ICI response in human trials, outperforming current theranostic biomarkers. Based on these data, we propose to expand and refine the QTL map for ICI response in the MC38 model with a focus on defining the genetic mechanisms for the epistatic interactions (Aim 1). In Aim 2, we will challenge the three top candidate genes, Csf2rb and Il2rb (mChr15) and Il10ra (mChr9), with available recombinant cytokines and blocking antibodies towards predicted changes in aPD1 response. These perturbations will allow us to further map the genetic network that establishes the optimal tumor immune microenvironment. We will then expand our studies by mapping the QTLs associated in the murine mammary cancer model AT3 (Aim 3). We anticipate that new QTLs for response will be uncovered. Finally, in Aim 4, we will use the CCF1 tumors to optimize an innovative in vitro micro-organosphere (MOS) platform that encapsulates cells of the tumor microenvironment in a permeable microcapsule amenable to pharmacological intervention and cellular interrogation. Once optimized, we will test a panel of ICI drugs alone and in combination on both murine and human tumors. The success of the proposed work will not only identify potential new biomarkers and new targets for augmenting clinical ICI response, but also will establish a validated platform for the discovery of host genes involved in the response to any immune checkpoint inhibitor.

NIH Research Projects · FY 2026 · 2024-12

PROJECT SUMMARY/ABSTRACT Deafness is a significant health concern that affects more than 450 million people worldwide, and both environmental insults and genetic mutations are contributing factors. The auditory organ in the inner ear is composed of sensory hair cells that detect and transmit sound vibrations. Hair cells are innervated by bipolar spiral ganglion neurons (SGNs) that relay auditory signals from the hair cells to the brainstem. Damage causing SGN degeneration or affecting SGN projections or synapses with hair cells leads to hearing loss. Mitochondrial dysfunction is a common cause of pathologies targeting organs that require large amounts of energy to perform optimally, such as neurons. Nuclear DNA mutations in genes encoding mitochondrial proteins can affect mitochondrial biogenesis or energy metabolism pathway, affecting auditory function in either case. A focus of this study, variants in the mitochondrial ACO2 protein have been associated with optic nerve atrophy, seizures, and hearing loss in young children. ACO2 is a nuclear gene that produces aconitase hydratase 2, an early enzyme in the tricarboxylic acid cycle. Unfortunately, cellular or mechanistic insight into ACO2-related conditions is lacking in part due to the absence of adequate animal models. We identified a spontaneous recessive R56L amino acid substitution in the mouse Aco2 gene based on circling behavior. Homozygotes are viable but show early-onset progressive hearing loss at 1 month of age followed by severe SGN loss at 2 months. I propose that the ACO2 R56L protein variant results in SGN apoptosis due to mitochondrial dysfunction, explaining early-onset hearing loss. Aim 1 of this proposal will track SGN defects in the Aco2 R56L mutants from developmental stages to young adults. Aim 2 will investigate how the R56L mutation affects the dose and localization of the ACO2 protein, and establish how SGN mitochondria number and distribution is altered in time in mutants. Aim 3 will attempt to rescue mitochondrial defects and loss of SGNs in mutants via metabolic supplementation. This will first be tested in organ culture prior to exploiting these results in live mice to improve auditory function. The proposed research has the potential to help develop realistic treatments to prevent or rescue hearing loss in ACO2 patients. Moreover, the new Aco2 R56L mouse strain will be invaluable to model other neurological disorders in ACO2 patients, including optic atrophy.

NIH Research Projects · FY 2025 · 2024-12

PROJECT SUMMARY Recent research advances support the significant role of vascular factors in neurodegeneration, cognitive decline, and dementia, including Alzheimer's disease (AD) and AD-related dementias (ADRDs). These vascular contributions encompass issues that include small vessel disease, cerebral amyloid angiopathy (CAA), and blood-brain barrier dysfunction. Conventionally, dementia resulting from vascular problems was labeled as vascular dementia, while AD was linked to neurodegeneration due to amyloid plaques and neurofibrillary tangles. However, recent data indicate that these conditions form a spectrum which includes mixed etiology dementias, where multiple brain pathologies coexist. Efforts to elucidate and understand the causal changes that underlie the vascular contributions to cognitive decline and dementia are being advanced by the use of genetic and biomarker studies in humans and model organisms, aided by implementation of new multi-omic technologies. However, despite the growing importance and relevance of this area of biomedical research, there is currently a lack of specialized conferences that address this topic and that provide a forum for sharing of advances and challenges – in particular one focused on training the next generation of scientists. To address this gap, we propose an intensive, trainee-focused, 4-day conference in 2025 at The Jackson Laboratory (JAX) in Bar Harbor, ME. This conference, entitled "Vascular Contributions to Cognitive Impairment and Dementia (VCID)", will leverage participation by a variety of NIH-funded consortia to bring together nearly 100 scientists, spanning career stages, from the fields of vascular biology, neurodegeneration, ADRD genetics, and computational biology, as well as provide a virtual attendance option for those that cannot or choose not to attend in person. This interdisciplinary event aims to foster data and model dissemination, networking, training, career development, and collaboration through seminars, workshops, discussions, and group activities. These objectives are facilitated by the residential nature of the JAX Highseas Conference Center. To achieve these objectives, we propose the following Specific Aims: Aim 1. Organize an interdisciplinary conference and interactive workshops on VCID. Aim 2. Promote interactions to foster collaborative research and career advancement. Aim 3. Foster the recruitment and development of diverse junior investigators in the VCID research field.

NIH Research Projects · FY 2024 · 2024-09

Alzheimer’s disease (AD), most common form of dementia, affects over 55 million people worldwide. Numbers are expected to double every 20 years unless improved treatments are developed. AD is characterized by amyloid plaques and neurofibrillary tangles of tau, but recently approved treatments targeting amyloid deposition show limited effectiveness. Therefore, it is critical that treatments are developed that target multiple aspects of AD and related dementias (ADRD). Small vessel cerebrovascular disease is now thought to contribute to many ADRD cases. However, no treatments that target cerebrovascular health in ADRD are available. Therefore, in response to RFA-NS-24-027 (VCID Center Without Walls [CWOW] for Understanding and Leveraging Small Vessel Cerebrovascular Disease Mechanisms in ADRD), we propose to establish The Jackson Laboratory (JAX)/Emory University/Columbia University/Rush University VCID CWOW to prioritize novel therapeutic targets for vascular contributions to cognitive impairment and dementia (VCID). We will focus on understanding factors mediating cerebral amyloid angiopathy (CAA: the deposition of amyloid in small vessels), including interactions with tau pathology and APOE genotype. APOE4 is the greatest genetic risk factor for ADRD and accelerates CAA. Despite the prevalence of CAA, the molecules that mediate its development remain to be elucidated. We hypothesize that targeting CAA, and the underlying cerebrovascular dysfunction and BBB breakdown, will prevent or slow neurodegeneration and cognitive decline in ADRD. To test our hypothesis, we will leverage expertise and resources available to the JAX/Emory/Columbia/Rush VCID CWOW. These include human samples and associated deep clinical data from the Religious Orders Study/Memory Aging Project (ROSMAP), a wealth of multi-omic data generated as part of the Accelerated Medicines Partnerships (AMP)- AD, and novel ADRD mouse models generated as part of Model Organism Development and Evaluation for Late-Onset AD (MODEL-AD). Our preliminary analyses have identified putative CAA-associated targets that are dependent on or independent of APOE4. However, small vessel-specific multi-omic datasets are lacking, hindering our sensitivity to identify cerebrovascular-specific mediators of CAA. Therefore, we will first generate and analyze vessel-enriched proteomic and single-nucleus RNA-seq datasets from ROSMAP samples showing variation in cognitive status, amyloid and tau pathology, CAA, and APOE genotype (Aim 1). We will then deeply phenotype our novel ADRD mouse model panel that shows a similar variation in CAA, APOE genotype, and tau pathology to that in the ROSMAP human samples. We will perform cognitive exams, magnetic resonance imaging, fluid biomarkers, whole-brain CAA mapping, and bulk and vessel-enriched proteomics (Aim 2). Finally, we will perform spatial transcriptomics and proteomics in human and mouse samples and integrate all data to prioritize novel vascular-specific candidates. The most promising of these will be validated in our mouse models (Aim 3). All data and resources will be made available through the AD Knowledge Portal and the JAX repository.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT Congenital Diaphragmatic Hernia (CDH) is a common and severe structural birth defect arising in 1 out of 3000 live births, accounting for 8% of all congenital anomalies and 1-2% of infant mortality. The high mortality rates are due to the abnormal lung hypoplasia and pulmonary hypertension that accompanies the herniation of the diaphragm muscle. The genetic etiology of CDH is highly heterogeneous and our understanding of the anomaly is incomplete. Although whole exome and whole genome sequencing has identified more than 100 candidate genes, only ~25 have been validated with reproducibility, and we estimate that many additional CDH genes and alleles are yet to be discovered. Animal model validation of these discoveries are often lacking, which is due, in part, to the fact that the diaphragm is a mammalian-specific structure. The generation and characterization of germline mouse models is costly and time-consuming, and therefore typically incompatible with the need to screen many candidate genes and variants. The high efficiency of CRISPR/Cas9 genome editing, and the appearance of phenotypes in founder animals has allowed us to develop a platform that can reduce the time for validation from more than one year, to 2-3 months. The overarching goal of this proposal is to identify novel genes and variants that are associated with human CDH and to validate these discoveries in the mouse. We will use genome data from our large, well-characterized and longitudinally studied CDH cohort to identify novel genes and variants in patients with CDH, and prioritize these genes and variants for validation in mouse models. We will screen prioritized variants using our high-throughput mouse F0 platform and select a subset of hits for germline modeling and more extensive characterization. Together, these studies will advance our understanding of the genetics and mechanisms underlying CDH, improve diagnostic tools for patients, and pave the way for the future development of therapeutics aimed at improving lung maturation and function.

NIH Research Projects · FY 2025 · 2024-09

PROJECT SUMMARY/ABSTRACT The human genome and that of its experimental surrogate, the mouse, contain a haploid DNA content of approximately three gigabase pairs (3 X 109 bp) and an estimated 20,000 genes. Interrogating this complex landscape of genes and developing community resources around them have traditionally involved modifying the genome on a gene-by-gene and resource-by-resource basis with tremendous effort and at great cost. For example, to create an animal resource containing a knockout of each mouse gene, the International Mouse Knockout Consortium has had to target each of 20,000 unique sequences at a combined cost of close to $1 billion (USD). Contributing to the funding and labor burden is the fact that each experiment is dependent upon the technically challenging and labor-intensive microinjection of DNAs into fertilized oocytes, or embryonic stem cells into early developmental stage mouse blastocysts. Creating even a second, new genome-wide resource based on each of 20,000 genes could conceivably require repeating the costly process yet again. To move beyond the one-by-one approach for developing genome-wide resources, outlined/envisioned in this proposal, is a powerful combinatorial technology to sequentially marry functional DNA sequences (encoding, for example, reporters, recombinases, cell-ablating toxins, protein interaction domains, and nucleases) to each of thousands of unique mouse genes already marked with a uniform (lacZ-) sequence tag. The potential for a breakthrough in genome analysis studies at the heart of the proposal comes from the combination of two powerful and proven technologies — first, Prime Editing, a CRISPR technology employing Reverse Transcriptase to insert DNA sequences at points of DNA modification; and second, expression of Prime Editing machinery directly from the genome (rather than administering it exogenously). The key aspect of the proposed experiments is to exploit the exquisite specificity of Prime Editing to guide the reverse transcription of pegRNA edits/cargoes to multiple, specific, previously (e.g., lacZ-) tagged loci. Importantly, successful Prime Editing from specific pegRNAs will afford the opportunity to distribute novel functional sequences to thousands of mouse genes without any need for the costly, time-consuming microinjection of zygotes or embryonic stem cells. Full implementation of such as system has the potential to accelerate the development, increase the number, and decrease the cost of mouse genome-wide resources by orders of magnitude, and to move strain development efforts from a gene-by-gene method to a more massively parallel approach. The combinatorial nature of the technology and the adaptability of the system to incorporate the latest in new technological developments will allow a longstanding contribution to human health.