Download or read book Therapeutic Gene Correction Strategies Based on CRISPR Systems or Other Engineered Site specific Nucleases written by Ayal Hendel and published by Frontiers Media SA. This book was released on 2023-05-16 with total page 157 pages. Available in PDF, EPUB and Kindle. Book excerpt:

Download or read book Targeted Genome Editing Using Site Specific Nucleases written by Takashi Yamamoto and published by Springer. This book was released on 2015-01-05 with total page 206 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases. Applications of genome editing tools are then described in detail, in iPS cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Tools that are mentioned include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, all of which have received much attention in recent years as breakthrough technologies. Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. Various genome modifications have proven successful, including gene knockout and knock-in experiments with targeting vectors and chromosomal editing. Genome editing technologies hold great promise for the future, for example in biomedical research, clinical medicine, and generation of crops and livestock with desirable traits. A wide range of readers will find this book interesting, and with its focus on applications in a variety of organisms and cells, the book will be valuable for life scientists in all fields.

Download or read book CRISPR Cas Systems written by Rodolphe Barrangou and published by Springer Science & Business Media. This book was released on 2012-12-13 with total page 300 pages. Available in PDF, EPUB and Kindle. Book excerpt: CRISPR/Cas is a recently described defense system that protects bacteria and archaea against invasion by mobile genetic elements such as viruses and plasmids. A wide spectrum of distinct CRISPR/Cas systems has been identified in at least half of the available prokaryotic genomes. On-going structural and functional analyses have resulted in a far greater insight into the functions and possible applications of these systems, although many secrets remain to be discovered. In this book, experts summarize the state of the art in this exciting field.

Download or read book The Use of CRISPR cas9 ZFNs TALENs in Generating Site Specific Genome Alterations written by and published by Academic Press. This book was released on 2014-11-04 with total page 594 pages. Available in PDF, EPUB and Kindle. Book excerpt: This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers recent research and methods development for changing the DNA sequence within the genomes of cells and organisms. Focusing on enzymes that generate double-strand breaks in DNA, the chapters describe use of molecular tools to introduce or delete genetic information at specific sites in the genomes of animal, plant and bacterial cells. Continues the legacy of this premier serial with quality chapters authored by leaders in the field Covers research methods in biomineralization science Contains sections on such topics as genome editing, genome engineering, CRISPR, Cas9, TALEN and zinc finger nuclease

Download or read book CRISPR Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases written by Luis María Vaschetto and published by CRC Press. This book was released on 2022-01-31 with total page 315 pages. Available in PDF, EPUB and Kindle. Book excerpt: The CRISPR-Cas9 genome-editing system is creating a revolution in the science world. In the laboratory, CRISPR-Cas9 can efficiently be used to target specific genes, correct mutations and regulate gene expression of a wide array of cells and organisms, including human cells. CRISPR-/Cas9 Based Genome Editing for Treating Genetic Disorders and Diseases is a unique reading material for college students, academicians, and other health professionals interested in learning about the broad range of applications of CRISPR/Cas9 genetic scissors. Some topics included in this book are: the role of the CRISPR/Cas9 system in neuroscience, gene therapy, epigenome editing, genome mapping, cancer, virus infection control strategies, regulatory challenges and bioethical considerations.

Download or read book Human Genome Editing written by National Academies of Sciences, Engineering, and Medicine and published by National Academies Press. This book was released on 2017-08-13 with total page 329 pages. Available in PDF, EPUB and Kindle. Book excerpt: Genome editing is a powerful new tool for making precise alterations to an organism's genetic material. Recent scientific advances have made genome editing more efficient, precise, and flexible than ever before. These advances have spurred an explosion of interest from around the globe in the possible ways in which genome editing can improve human health. The speed at which these technologies are being developed and applied has led many policymakers and stakeholders to express concern about whether appropriate systems are in place to govern these technologies and how and when the public should be engaged in these decisions. Human Genome Editing considers important questions about the human application of genome editing including: balancing potential benefits with unintended risks, governing the use of genome editing, incorporating societal values into clinical applications and policy decisions, and respecting the inevitable differences across nations and cultures that will shape how and whether to use these new technologies. This report proposes criteria for heritable germline editing, provides conclusions on the crucial need for public education and engagement, and presents 7 general principles for the governance of human genome editing.

Download or read book Genome Engineering via CRISPR Cas9 System written by Vijai Singh and published by Academic Press. This book was released on 2020-02-21 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Genome Engineering via CRISPR-Cas9 Systems presents a compilation of chapters from eminent scientists from across the globe who have established expertise in working with CRISPR-Cas9 systems. Currently, targeted genome engineering is a key technology for basic science, biomedical and industrial applications due to the relative simplicity to which they can be designed, used and applied. However, it is not easy to find relevant information gathered in a single source. The book contains a wide range of applications of CRISPR in research of bacteria, virus, algae, plant and mammalian and also discusses the modeling of drosophila, zebra fish and protozoan, among others. Other topics covered include diagnosis, sensor and therapeutic applications, as well as ethical and regulatory issues. This book is a valuable source not only for beginners in genome engineering, but also researchers, clinicians, stakeholders, policy makers, and practitioners interested in the potential of CRISPR-Cas9 in several fields.

Download or read book Reprogramming the Genome CRISPR Cas based Human Disease Therapy written by and published by Academic Press. This book was released on 2021-06-12 with total page 394 pages. Available in PDF, EPUB and Kindle. Book excerpt: Reprogramming the Genome: CRISPR-Cas-based Human Disease Therapy, presents the collation of chapters written by eminent scientists worldwide. CRISPR-Cas9 is a key technology for targeted genome editing and regulation in a number of organisms including mammalian cells. It is a rapid, simple, and cost-effective solution. CRISPR-Cas system has recently gained much scientific and public attention. This volume covers CRISPR-Cas9 based mammalian genome editing, creating disease models, cancer therapy, neurological, heredity, blood disorders, defective gene correction, stem cells therapy, epigenetic modifications, patents, ethics, biosafety and regulatory issues challenges and opportunities. This book is a key source of information on mammalian genome editing available in a single volume. This book will be useful for beginners in mammalian genome editing and also students, researchers, scientists, policymakers, clinicians and stakeholders interested in genome editing in several areas. Offers basic understanding and a clear picture of mammalian genome editing through CRISPR-Cas systems Discusses how to create mammalian disease models, stem cell modification, epigenetic modifications, correction of defective gene in blood disorders, heredity, neurological disorders and many more Discusses the application of CRISPR-Cas9 systems in basic sciences, biomedicine, molecular biology, translational sciences, neurobiology, neurology, cancer, stem cells, and many more

Download or read book Precision Medicine CRISPR and Genome Engineering written by Stephen H. Tsang and published by Springer. This book was released on 2017-11-11 with total page 180 pages. Available in PDF, EPUB and Kindle. Book excerpt: This book presents descriptive overviews of gene editing strategies across multiple species while also offering in-depth insight on complex cases of application in the field of tissue engineering and regenerative medicine. Chapters feature contributions from leaders in stem cell therapy and biology, providing a comprehensive view of the application of gene therapy in numerous fields with an emphasis on ophthalmology, stem cells, and agriculture. The book also highlights recent major technological advances, including ZFN, TALEN, and CRISPR. Precision Medicine, CRISPR, and Genome Engineering is part of the highly successful Advances in Experimental Medicine and Biology series. It is an indispensable resource for researchers and students in genetics as well as clinicians.

Download or read book Artificial Nucleases written by Marina A. Zenkova and published by Springer Science & Business Media. This book was released on 2004-01-07 with total page 330 pages. Available in PDF, EPUB and Kindle. Book excerpt: The development of agents capable of cleaving RNA and DNA has attracted considerable attention from researchers in the last few years, because of the immediate and very important applications they can find in the emerging fields of biotechnology and pharmacology. There are essentially two classes of these agents - nucleases that occur naturally inside cells and synthetically produced artificial nucleases. The first class includes protein enzyme nucle ases and catalytic RNA structured ribozymes that perform cleavage of the phosphodiester bonds in nucleic acids according to a hydrolytic pathway in the course of different biochemical processes in the cell. A different pathway is used by some antibiotics which cleave DNA via redox-based mechanisms resulting in oxidative damage of nucleotide units and breakage of the DNA backbone. The above molecules are indispensable tools for manipulating nucleic acids and processing RNA; DNA-cleaving antibiotics and cytotoxic ribonucleases have demonstrated utility as chemotherapeutic agents. The second class, artificial nucleases, are rationally designed to imitate the active centers of natural enzymes by simple structures possessing minimal sets of the most important characteristics that are essential for catalysis. A dif ferent approach, in vitro selection, was also used to create artificial RNA and DNA enzymes capable of cleaving RNA. Being less efficient and specific as compared to the natural enzymes, the primitive mimics are smaller and robust and can function in a broad range of conditions.

Download or read book Genome Editing with CRISPR Cas9 to Study and Treat Primary Immune Regulatory Disorders written by Esmond Lee and published by . This book was released on 2022 with total page pages. Available in PDF, EPUB and Kindle. Book excerpt: For those living with genetic disease, the advent of gene therapy represents an end to years of repeated treatment and daily disease management. Using gene editing tools, patient cells can be modified to provide long term therapeutic benefit that cures or mitigates disease. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology allows us to make precise genetic changes to cells for therapeutic benefit. During CRISPR gene editing, single guide RNA (sgRNA) directs Cas9 nuclease to make a double-stranded cut to DNA at a precise location in the genome. A DNA repair cassette delivered by recombinant adeno-associated virus (rAAV) is then inserted at the cut site via homologous recombination. This repair cassette can be designed to contain a therapeutic gene which can be expressed at physiological levels, under the control of endogenous regulatory elements. Monogenic disorders of the blood and immune system are severe diseases with limited therapeutic options. While allogeneic hematopoietic stem cell transplantations (HSCT) have historically been used to treat blood and immune disorders, the rise of CRISPR/Cas9 technology has enabled us to modify a patient's own stem cells for autologous transplantation, thereby circumventing the challenges associated with rejection. Hematopoietic stem and progenitor cells (HSPCs) are particularly amenable to gene therapy because we can isolate, culture, and modify them ex vivo. This dissertation explores CRISPR/Cas9 based gene editing as a new therapeutic modality for two primary immune regulatory disorders caused by mutations in the FOXP3 and IL-10 genes, respectively. The goal of this therapeutic modality is to engraft gene edited patient HSPCs to enable healthy hematopoietic reconstitution, curing the disease. The gene editing strategy was validated through assaying the phenotype and function of edited primary T cells; the engraftment and differentiation potential of edited HSPCs were subsequently confirmed in the NSG-SGM3 humanized mouse model. Further, CRISPR/Cas9 knockout experiments elucidated the mechanisms of dysregulation in specific immune subsets, advancing our understanding of the role of FOXP3 and IL-10 in health and disease. Another aspect of bringing cell and gene therapies to the clinic is the complexity and cost of manufacturing. An understated factor that could expedite patient access to gene therapies is robust and cost-efficient manufacturing processes. The final segment of this dissertation applies a regression-based approach to understand key factors influencing gene editing frequencies in CD4+ T cells. Editing frequencies across varying guide and rAAV concentrations were linked to a cost of raw materials analysis model to provide information on the cost drivers of gene targeting and to predict cost-optimal editing conditions for cell therapy manufacturing.

Download or read book Gene Editing in Plants written by and published by Academic Press. This book was released on 2017-07-14 with total page 266 pages. Available in PDF, EPUB and Kindle. Book excerpt: Gene Editing in Plants, Volume 149 aims to provide the reader with an up-to-date survey of cutting-edge research with gene editing tools and an overview of the implications of this research on the nutritional quality of fruits, vegetables and grains. New chapters in the updated volume include topics relating to Genome Engineering and Agriculture: Opportunities and Challenges, the Use of CRISPR/Cas9 for Crop Improvement in Maize and Soybean, the Use of Zinc-Finger Nucleases for Crop Improvement, Gene Editing in Polyploid Crops: Wheat, Camelina, Canola, Potato, Cotton, Peanut, Sugar Cane, and Citrus, and Gene Editing With TALEN and CRISPR/Cas in Rice. This ongoing serial contain contributions from leading scientists and researchers in the field of gene editing in plants who describe the results of their own research in this rapidly expanding area of science. Shows the importance of revolutionary gene editing technology on plant biology research and its application to agricultural production Provides insight into what may lie ahead in this rapidly expanding area of plant research and development Contains contributions from major leaders in the field of plant gene editing

Download or read book CRISPR Cas Enzymes written by and published by Academic Press. This book was released on 2019-01-25 with total page 451 pages. Available in PDF, EPUB and Kindle. Book excerpt: CRISPR-Cas Enzymes, Volume 616, the latest release in the Methods in Enzymology series, continues the legacy of this premier serial with quality chapters authored by leaders in the field. Topics covered in this release include CRISPR bioinformatics, A method for one-step assembly of Class 2 CRISPR arrays, Biochemical reconstitution and structural analysis of ribonucleoprotein complexes in Type I-E CRISPR-Cas systems, Mechanistic dissection of the CRISPR interference pathway in Type I-E CRISPR-Cas system, Site-specific fluorescent labeling of individual proteins within CRISPR complexes, Fluorescence-based methods for measuring target interference by CRISPR-Cas systems, Native State Structural Characterization of CRISRP Associated Complexes using Mass Spectrometry, and more. Provides the authority and expertise of leading contributors from an international board of authors Presents the latest release in the Methods in Enzymology series Updated release includes the latest information on the CRISPR-Cas Enzymes

Download or read book Genome Editing and Engineering written by Krishnarao Appasani and published by Cambridge University Press. This book was released on 2018-08-23 with total page 535 pages. Available in PDF, EPUB and Kindle. Book excerpt: Recent advances in genome editing tools using endonucleases such as TALENs, ZFNs, and CRISPRs, combined with genomic engineering technologies, have opened up a wide range of opportunities from applications in the basic sciences and disease biology research, to the potential for clinical applications and the development of new diagnostic tools. This complete guide to endonuclease-based genomic engineering gives readers a thorough understanding of this rapidly expanding field. Chapters cover the discovery, basic science, and application of these techniques, focusing particularly on their potential relevance to the treatment of cancer, and cardiovascular and immunological disease. The final section discusses the legal and ethical issues which accompany the technology. Providing authoritative coverage of the potential that genome editing and engineering have, this is an ideal reference for researchers and graduate students and those working in the biotechnology and pharmaceutical industries, as well as in a clinical setting.

Download or read book Improving Nuclease Mediated Gene Editing Outcomes in Human Hematopoietic Stem Cells written by Anastasia Lomova and published by . This book was released on 2019 with total page 183 pages. Available in PDF, EPUB and Kindle. Book excerpt: Autologous hematopoietic stem cell (HSC) transplantation, combined with gene editing, could provide an ideal therapeutic option for the treatment of congenital blood diseases, such as hemoglobinopathies, primary immune deficiencies, and storage disorders. Gene editing relies on site-specific induction of a double stranded break (DSB) by targeted nucleases (such as Zinc Finger Nucleases (ZFNs) or CRISPR/Cas9 system), and subsequent gene correction using endogenous cellular repair mechanisms. The two main competing pathways to repair the break are non-homologous end joining (NHEJ), an often-imprecise pathway which can result in insertions and deletions (indels), or accurate homology-directed repair (HDR) pathway which uses a homologous donor template to seamlessly repair the break and incorporate the desired changes. For certain diseases, where a knockout of a gene can result in therapeutic benefit, repair by NHEJ pathway may be favorable. However, for conditions where disruption of a gene can result in an even more severe phenotype than the original disease (such as sickle cell anemia), repair via HDR pathway is critical. Despite advances in nuclease technologies and the ability to efficiently achieve high frequency of site-specific gene disruption, the current progress to reach clinically relevant levels of precise HDR-mediated repair still remains elusive. Therefore, our translational goal is to improve the gene editing outcomes in HSCs, specifically, increase HDR and decrease NHEJ levels, which will be beneficial for treating many diseases of the blood. This dissertation aims to identify the hindrances that limit efficient HDR-mediated editing in HSCs, and investigates several approaches to address these impediments. Our results indicate that one major reason for low gene correction in HSCs is their heightened susceptibility to cell toxicity resulting from the electroporation of the nuclease and homologous donor template. We demonstrate that co-electroporation of mRNA encoding the anti-apoptotic protein BCL2 with gene editing reagents significantly ameliorates the cytotoxicity and increases the yield of gene-corrected HSCs. Next, we show that cell cycle-dependent control of nuclease activity and DNA repair pathways can influence gene editing outcomes to favor the precise DNA modification (HDR) over faulty repair events (NHEJ) in human HSCs. By using a modified version of Cas9 protein with reduced nuclease activity in G1 phase of cell cycle, when HDR cannot occur, and transiently increasing the proportion of cells in HDR-preferred phases (S/G2), we achieve a 4-fold improvement in HDR/NHEJ ratio over the control condition in vitro, and a significant improvement in long-term gene-modified engrafted cells after xenotransplantation of edited human HSCs into immune-deficient mice. Finally, we investigate what cellular elements govern the DNA repair pathway choice and how they can be exploited to shift the balance toward HDR from NHEJ. We test the effects of manipulating the expression levels of several DNA repair factors, that are presumed to be important for pathway choice and progression, on HDR and NHEJ levels in K562 cell line and primary human hematopoietic stem and progenitor cells (HSPCs). Interestingly, we observe differential effects of DNA repair factor manipulation on gene editing outcomes dependent upon the delivery method employed and the types of cells used. These strategies for improving gene editing outcomes in human HSCs have important implications for the field of gene therapy as a whole, and can be applicable to diseases where increased HDR/NHEJ ratio is critical for therapeutic success.

Download or read book Enhancing the Efficiency of CRISPR Cas9 Precise Gene Editing for Cystic Fibrosis Gene Therapy written by Kyle Seigel and published by . This book was released on 2018 with total page 0 pages. Available in PDF, EPUB and Kindle. Book excerpt: Cystic fibrosis (CF) is the most common cause of chronic obstructive lung disease in children and young adults, yet there is no cure for this disease. Intimations of curative strategies have emerged from gene therapy, but these approaches have failed in clinical trials due to insufficient safety and efficacy profiles. The molecular technology necessary to overcome preeminent challenges in gene therapy may reside in programmable nucleases - namely CRISPR/Cas9 - which allow for site-specific genomic integration of therapeutic transgenes. However, the clinical utility of these technologies is limited by inherently low efficiencies. Here, we use flow cytometry and PCR assays to demonstrate the efficacy of perturbing DNA repair to overcome such limitations. We demonstrate that overexpressing CtIP and EXO1-4D can enhance the efficiency of CRISPR/Cas9-directed transgene integration by ~3- and ~6-fold, respectively. These discoveries may provide necessary impetus for translating gene therapies into clinical realities for genetic diseases such as CF.

Download or read book Engineering Efficient and Safe in Situ Genome Regulation Via CRISPR Cas9 for Enabling Gene Therapies written by Ana Maria Moreno and published by . This book was released on 2019 with total page 175 pages. Available in PDF, EPUB and Kindle. Book excerpt: While the genetic and pathogenic basis of human diseases continues to grow, translation is currently bottlenecked by lack of tools and technologies to administer and evaluate corresponding gene-based therapeutics. Consequently, development of safe and efficient in vivo gene transfer platforms, coupled with emerging genome and epigenome engineering tools, will transform our ability to target a range of human diseases. In this regard, the holy grail of in vivo genome engineering is the ability to achieve the trifecta of: 1) efficient and safe delivery; 2) temporally regulatable and tunable payload delivery; and 3) immune stealth to minimize dosage & enable re-administration of nucleic acid or protein therapeutics. Towards this, the objective of this dissertation was to develop a platform to enable efficacious in vivo genome and epigenome engineering with a focus on enabling in situ therapeutic efficacy. The studies in this dissertation are independent bodies of work that explore the optimization and engineering of CRISPR-Cas9 systems to bring these one step closer to their eventual translation into the clinic. Towards these, I first developed a robust and generalizable platform for in situ genome editing and regulation via AAV CRISPR-Cas9. Towards this, I utilized split-Cas9 systems to develop a modular adeno-associated viral (AAV) vector platform for CRISPR-Cas9 delivery to enable the full spectrum of targeted in situ gene regulation functionalities, demonstrating robust transcriptional repression (up to 80%) and activation (up to 6-fold) of target genes in cell culture and mice. We also applied our platform for targeted in vivo gene-repression-mediated gene therapy for retinitis pigmentosa. Specifically, we engineered targeted repression of Nrl, a master regulator of rod photoreceptor determination, and demonstrated Nrl knockdown mediates in situ reprogramming of rod cells into cone-like cells that are resistant to retinitis pigmentosa-specific mutations, with concomitant prevention of secondary cone loss. Furthermore, we benchmarked our results from Nrl knockdown with those from in vivo Nrl knockout via gene editing. Taken together, our AAV-CRISPR-Cas9 platform for in vivo epigenome engineering enables a robust approach to target disease in a genomically scarless and potentially reversible manner. Additionally, this is the first time that the utility of AAV-KRAB-dCas9 mediated in situ gene repression in the context of gene therapy was demonstrated (Moreno et al., WIREs Systems Biology and Medicine, 2017; Moreno et al., Molecular Therapy, 2018). Next, I focused on addressing, arguably the most important hurdle for CRISPR-Cas based gene therapies, which is the interaction of these non-host derived systems with the adaptive immune system which can lead to neutralization by circulating antibodies and clearance of treated cells by cytotoxic T-lymphocytes. To address this issue, I proposed a new approach: sequential use of orthologous proteins that are orthogonal in immune space. This would, in principle, allow for repeated treatments by thus chosen orthologs without reduced efficacy due to lack of immune cross-reactivity among the proteins. To explore and validate this concept we chose 284 DNA targeting and 84 RNA targeting CRISPR effectors (including Cas9, Cpf1/Cas12a, and Cas13a, b, and c), and 167 Adeno-associated virus (AAV) capsid protein orthologs and developed a pipeline to compare total sequence similarity as well as predicted binding to class I and class II Major Histocompatibility Complex (MHC) proteins. Our MHC binding predictions revealed wide diversity among the set of DNA-targeting Cas orthologs, with 79% of pairs predicted not to elicit cross-reacting immune responses, while no global immune orthogonality among AAV serotypes was observed. We validated the computationally predicted immune orthogonality among three important Cas9 orthologs, from S. pyogenes, S. aureus, and C. jejuni observing cross-reacting antibodies against AAV but not Cas9 orthologs in sera from immunized mice. Finally, to demonstrate the efficacy of multiple dosing with immune orthogonal orthologs, we delivered AAV-Cas9 targeting PCSK9 into BALB/c mice previously immunized against the AAV vector and/or the Cas9 payload, demonstrating that editing efficiency is compromised by immune recognition of either the AAV or Cas9, but, importantly, this effect is abrogated when using immune orthogonal orthologs. Moving forward, we anticipate this framework can be applied to prescribe sequential transient regimens of immune orthogonal protein therapeutics to circumvent pre-existing or induced immunity, and eventually, to rationally engineer immune orthogonality among protein orthologs. (Moreno, Palmer et al., Nature Biomedical Engineering, in press, 2019). Lastly, I then proceeded to integrate the advances accomplished in the previous chapters to enable pain management via in situ genome repression. In the US and worldwide, pain is a leading cause of disability, which leads to a diminished quality of life. Patients have come to routinely expect pharmacological management, with the prevalent aggressive approach for managing pain states being based on opiates. While the utility of opiates has made them a mainstay of pain management, there are at least four key reasons supporting the need for new and alternative pain therapeutics: limited efficacy, abuse potential, tolerance after continued exposure, and an enhancement of post-wound pain states. Despite decades of research, broad-acting, longer-lived, non-addictive, and effective drugs for chronic pain remain elusive. Notably, genetic studies have correlated a hereditary loss-of-function mutation in a human Na+ channel isoform--NaV1.7--with a rare genetic disorder, Congenital Insensitivity to Pain (CIP), which leads to insensitivity to pain without other neurodevelopmental alterations. While an excellent target, the creation of blockers for this site has not led yet to an efficient and safe drug, due to their lack of specificity, leading to unwanted side-effects. Taking advantage of this druggable target in the human genome, the aim of this work was to develop a novel therapeutic regiment via in situ NaV1.7 repression to regulate the development and maintenance of impending chronic pain states. In this regard, I demonstrated robust in vitro repression using two epigenome engineering tools (KRAB-CRISPR-Cas9 and KRAB-Zinc-Fingers) with ~71% and ~88% repression respectively. To enable pain management, I injected mice intrathecally using the constructs with the highest in vitro efficacy and demonstrated robust NaV1.7 repression with a significant improvement in pain response in a carrageenan inflammatory pain model. I demonstrated a 133% improvement in paw withdrawal latency as compared to a negative control (mCherry) and 62% improvement over the positive control (Gabapentin, 100 mg/kg). Taken together, the use of these in situ engineering approaches could thus represent a viable replacement for opioids and a potential therapeutic approach that is tunable and reversible (Moreno et al., in preparation). Together, the advances in these bodies of work, which demonstrate efficacious in vivo delivery and gene editing/regulation is a significant step toward their implementation for gene therapeutic applications.