Browsing by Subject "CRISPR-Cas Systems"
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Item CRISPR and gene editing: one tool to rule them all(2017-05-09) Wolinetz, Carrie D.Advances in gene editing, particularly the development of CRISPR-cas9, have allowed for new applications of this technology, ranging from gene drives to development of new animal models for research. This emerging biotechnology is pushing the boundaries of science, even as it provides new and evolving challenges to our policy framework and oversight mechanisms. How do we ensure responsible and feasible oversight while not constraining scientific progress that expands our knowledge base and improves human health? What are the intersection points between new gene editing applications and the current policy landscape?Item The Design, Synthesis, and Evaluation of Zwitterionic and Cationic Lipids for In Vivo RNA Delivery and Non-Viral CRISPR/Cas Gene Editing(2018-03-05) Miller, Jason Brian; Ready, Joseph M.; Gao, Jinming; Tambar, Uttam; Siegwart, Daniel J.The delivery of nucleic acids is an emerging therapeutic modality in clinical development for the treatment of many genetic diseases. The use of RNA interference (RNAi) as a therapeutic is an exciting and rapidly developing field that offers a promising alternative to small molecule drugs for the treatment of dysregulatory diseases, including cancer. Small interfering RNA (siRNA) can be designed against any mRNA target, and upon loading into the RNA-induced silencing complex (RISC) can enable sequence-specific target recognition and degradation. Meanwhile, messenger RNA is currently being utilized for protein replacement therapy and for the development of vaccines by expressing viral antigens on dendritic cells. However, because RNA molecules are unable to passively diffuse across plasma membranes due to a high molecular weight (~13 kDa for siRNA, >300 kDa for mRNA), hydrophilicity and strong anionic charge, while also being unstable and highly immunogenic when injected systemically, nucleic acid therapeutics require carriers for effective delivery. To date, many successful carriers have been designed using amphiphilic lipid-like compounds containing amine-rich cores, but the challenges of efficient endosomal release and delivery to organs outside of the liver remain major hurdles in the field of RNA therapeutics. This dissertation reports the design, synthesis and characterization of two new classes of lipids with unique chemical structures and in vivo RNA delivery capabilities to the lung: zwitterionic amino lipids (ZALs) and cationic sulfonamide amino lipids (CSALs). ZALs contain an amine rich core, hydrophobic tails introduced via conjugate addition or epoxide opening, and a zwitterionic sulfobetaine head group. ZALs were designed with a combination of cationic and zwitterionic lipid properties, to help stabilize and effectively deliver long RNA molecules. A lead compound, ZA3-Ep10, was effective for in vivo messenger RNA delivery and the first reported demonstration of in vivo non-viral gene editing by delivering mRNA components encoding the CRISPR/Cas gene editing platform. CSALs contain a unique chemical scaffold containing an internal quaternary ammonium group and a sulfonamide linker. A rational investigation of structure-activity relationships revealed that CSALs containing an acetate sidearm, a dimethyl amino head group and higher hydrophobic content were effective in delivery siRNA to human cancer cells in vitro. CSALs also demonstrated lung localization upon systemic delivery in vivo while also demonstrating the ability to redirect liver targeting ionizable lipid nanoparticles to the lung. These new classes of materials demonstrate the importance of structural consideration in material design for the development of nucleic acid therapeutics, while also providing structural templates for developing carriers for effective delivery to tissues outside of the liver.Item Gene drives on the horizon: challenges in science, ethics, and governance(2017-12-12) Heitman, Elizabeth[Note: The slide presentation and video are not available from this event.] Since 2015, researchers using the gene editing technique CRISPR-Cas9 have developed laboratory-based proofs-of-concept for gene drives in yeast, fruit flies, mosquitoes, and mice that can efficiently introduce specific gene traits throughout a population. Most gene drive research to date has focused on controlling or altering organisms such as mosquitoes that transmit infectious diseases to humans, but gene drives have also been proposed as a way to address other complex and persistent problems in public health, agriculture, and conservation. This presentation will examine the science of gene drive research in non-human organisms and related challenges in ethics and governance. Reflecting on the work of the National Academies of Sciences' committee for which she served as co-chair, Dr. Heitman will describe the fundamental science of gene drives and the ethical concerns they raise, the role of public engagement in risk assessment, and the recommendations for responsible practices in gene drive research that the Committee made in its June 2016 report, Gene Drives on the Horizon.Item Identifying Novel Regulators of LIN28B Through a Genome-Wide CRISPR/Cas9 Screen(2017-01-17) Budhipramono, Albert; Hao, Zhu; Nguyen, LiemLIN28 is a family of RNA-binding proteins that are well-conserved across species. It is well-known to regulate developmental timing by inhibiting the biogenesis of the let-7 microRNAs. Numerous studies have shown that LIN28 is dysregulated in a wide spectrum of cancer types, especially pediatric cancers such as hepatoblastoma and Wilms' tumor. Our laboratory previously showed that reactivation of LIN28B, one of the two LIN28 homologs, is sufficient to drive liver cancer, and that LIN28B deletion is detrimental to tumor development. LIN28B exerts its oncogenic function by inhibiting the maturation of let-7 precursors, as well as directly binding to and enhancing translation of growth-promoting mRNA targets, such as members of the IGF2BP family. While our work and others established that LIN28B functions as an oncogene, the identity of factors that regulate LIN28B expression during normal development and cancer remains elusive. As LIN28B is a driver of oncogenesis in various cancers, understanding its regulation in the process of oncogenesis will help uncover novel therapeutic targets. Here, we show an original approach for identifying regulators of the human LIN28B gene utilizing the CRISPR/Cas9 genome engineering system. Traditional transgenic approaches to study gene function often fail to capture transcriptional regulation at distal promoter and enhancer sequences. Using the CRISPR/Cas9 system, we knocked a GFP reporter sequence into the endogenous locus of LIN28B in human cancer cell lines, engineering a fusion LIN28B-GFP protein. This approach is unique in that GFP expression will be altered not only by changes in regulation at the coding sequence, mRNA and protein levels, but also changes at distal regulatory sequences. To identify unknown regulators of LIN28B, we will perform a genome-wide CRISPR/Cas9-mediated knockout screen in human cells expressing the fusion LIN28B-GFP protein. Using a genome-scale library with 76,441 sgRNAs, we will knock out 19,114 genes individually and assess their effects on LIN28B levels by measuring GFP expression. sgRNAs that are enriched in the high GFP-expressing population suggest genes that normally function as inhibitors of LIN28B. On the other hand, sgRNAs that are depleted suggest activators of LIN28B. Through this screen, we hope to gain further insight into how LIN28B is regulated in normal development and cancer. Furthermore, identifying regulators of LIN28B can provide novel avenues for developing cancer therapeutics.Item Mechanisms of Genome Buffering and Cell Fate Coordination in Adult Tissue Homeostasis(2016-07-26) Tuladhar, Rubina; Amatruda, James F.; Scherer, Philipp; DeBerardinis, Ralph J.; Lum, LawrenceSelf-renewal competency of adult stem cells is essential for tissue homeostasis. The corruption of genes essential for genome preservation or for niche-stem cell interactions frequently results in loss of stem cell viability and disease. The two components of my thesis focus on understanding adult stem cell preservation - the integration of metabolism and intercellular communication mediated by the Wnt family of secreted signaling molecules, and epigenetic mechanisms that buffer the proteome against insertion/deletion (INDEL)-type genetic mutations. Wnt-mediated signaling is essential for embryogenesis and the maintenance of adult tissues. Lipidation of Wnt proteins by the acyltransferase Porcupine (Porcn) is crucial for secretory pathway exiting. Using chemically based approaches, I have demonstrated that Porcn active site features conserved across animals enforce ω-7 cis fatty acylation of Wnt proteins. Deviant acylation of a Wnt protein using an exogenously supplied trans fatty acid cripples its ability to traverse the secretory pathway due to a previously unappreciated stereoselectivity of the Wnt chaperone Wntless (WLS) for fatty acids. My findings provide a mechanistic account of chemical specificity observed in Porcn inhibitors, and delineate a universal mechanism for integrating communal cell fate decision-making with metabolic fitness. As part of my efforts to generate isogenic cells for the expression of LKB1, a tumor suppressor that regulates Wnt protein production, I encountered the emergence of foreign LKB1 proteins subsequent to the introduction of INDELs by the DNA editing enzyme CRISPR-Cas9. I demonstrate that these novel proteins are the products of: a) the installation of internal ribosomal entry sites (IRES), b) the induction of exon skipping due to compromised exon splicing enhancers (ESEs), and c) the conversion of pseudo-mRNAs to protein-coding mRNAs due to the unwanted elimination of premature termination codons. I propose that these molecular events serve as compensatory mechanisms employed by cells to restore proteome integrity in the face of INDEL-type challenges to the genome posed by pathogens and environmental mutagens. Taken together, these two projects will: a) delineate intervention strategies premised upon the attack of an universally conserved point of intersection between metabolism and cell-to-cell communication, b) facilitate the personalization of medicine, and c) accelerate tissue engineering initiatives.Item Prevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editing(2020-01-13) Zhang, Yu; Sadek, Hesham A.; Olson, Eric N.; Mendell, Joshua T.; Chen, ElizabethSkeletal muscle is one of the largest tissues in the human body and hence muscle diseases caused by genetic mutations have a profound and systemic impact on human health. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder, caused by mutations in the DMD gene on the X chromosome, which consists of 79 exons encoding dystrophin protein. Patients with DMD develop progressive muscle weakness and cardiomyopathy, and ultimately succumb to respiratory and cardiac failure in their mid-20s. The dystrophin gene was identified three decades ago and mutations in the DMD gene are well-characterized. However, there is no effective treatment for this debilitating disease. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) was first discovered as an adaptive immune system in bacteria and archaea for defending against phage infection. Recently, the CRISPR/Cas system has been applied for mammalian genome editing because it provides site-specific DNA double-stranded breaks with simplicity and precision. In this study, I demonstrate the feasibility of using CRISPR/Cpf1 to correct a DMD exon 48-50 out-of-frame deletion mutation in cardiomyocytes derived from patient induced pluripotent stem cells by exon skipping and exon reframing strategies. Next, I precisely correct a Dmd exon 23 nonsense mutation in mdx mouse by CRISPR/Cpf1-mediated germline editing. Furthermore, I apply CRISPR/Cas9-mediated post-natal genome editing to correct a Dmd exon 44 out-of-frame deletion mutation in a DMD mouse model. Finally, I develop an effective strategy to improve CRISPR/Cas9-mediated in vivo genome editing by packaging Cas9 nuclease in conventional single-stranded AAV and CRISPR single guide RNAs in double-stranded self-complementary AAV. This strategy significantly reduces the amount of AAV vector needed for therapeutic genome editing and enhances dystrophin restoration after delivery into a mouse model of DMD harboring an exon 44 deletion. These findings represent an important advancement toward therapeutic translation of genome editing technology for permeant correction of Duchenne muscular dystrophy.Item Prevention of Muscular Dystrophy in Mice by Gene Editing(2014-11-20) Long, Chengzu; Wang, Zhigao; Olson, Eric N.; Cobb, Melanie H.; Chen, Zhijian J.Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened lifespan, often along with breathing and heart complications. There is no effective treatment. RNA-guided nucleases-mediated genome editing, based on Type II CRISPR/Cas systems, offers a new approach to alter the genome. It can precisely remove a mutation in DNA, allowing the DNA repair mechanisms to replace it with a normal copy of the gene. The benefit of this over other gene therapy techniques is that it can permanently correct the 'defect' in a gene rather than just transiently adding a 'functional' one. We used CRISPR/Cas9-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germline of mdx mice, a model for DMD, and then monitored skeletal muscle and heart structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. Histological analysis of skeletal muscle and heart from these corrected mice showed absence of the dystrophic muscle phenotype and restoration of dystrophin expression. In addition, the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected stem cells and their contribution to regenerating muscle. Our experiments provide proof-of-concept that CRISPR/Cas9-mediated genomic editing can correct a causative germline mutation causing muscular dystrophy in a mouse model and prevent development of several characteristic features of the disease. With rapid technological advances of gene delivery systems and improvements to the CRISPR/Cas9 editing system, this strategy may allow correction of disease-causing mutations in the muscle tissue or iPSCs (induced pluripotent stem cells) from patients with genetic diseases.Item Re-engineering of Dendrimer-Based Lipid Nanoparticles for Efficient and Precise HDR-Mediated Gene Editing(August 2021) Farbiak, Lukas John; Lux, Jacques; Siegwart, Daniel J.; Corbin, Ian R.; Bachoo, Robert; Kim, Tae-KyungCRISPR/Cas gene editing is poised to transform the treatment of genetic diseases. However, limited progress has been made toward precise editing of DNA via Homology Directed Repair (HDR) that requires careful orchestration of complex steps. Rather, many reports of in vivo gene editing rely on an error-prone mechanism called Non-Homologous End Joining (NHEJ). While this pathway is effective for elimination of protein function via the introduction of insertions and deletions (Indels) into the genome, it presents little to no utility for correcting disease-causing mutations in DNA. As such, there is a pressing need to develop effective non-viral carriers capable of replacing the mutated DNA sequence with the corrected sequence via HDR. Currently, non-viral, in vivo gene editing techniques have been limited in their capacity to precisely correct mutations in DNA, with most examples yielding correction rates of less than 1%. Additionally, many delivery systems aimed at inducing HDR have consisted of multiple transfection vehicles, including virus, due to the diverse set of nucleic acid cargoes required for this process. However, techniques relying on viral vectors and/or separate carriers are nonoptimal due to potential immunogenicity and process dependence all three nucleic acids. This dissertation details the development of dendrimer-based lipid nanoparticles (dLNPs) for the encapsulation and delivery of multiple nucleic acids necessary for HDR: Cas9 mRNA, sgRNA, and a donor DNA template containing the correct nucleic acid sequence, as well as the optimization of intra-particle nucleic acid ratios for efficient induction of HDR in vivo. To assess in vivo HDR efficiency, we employed xenograft tumors consisting of BFP/GFP switchable HEK293 cells with a single Y66H amino acid mutation. Through systematically adjusting the individual internal ratios of Cas9 mRNA, sgRNA, and donor ssDNA, an optimal balance of components resulted in a HDR rate of greater than 20% in vivo. This is the first report of a completely non-viral, LNP-based, fully nucleic acid-mediated delivery system capable of inducing HDR. Due to the all-in-one simplicity and high efficacy, HDR dLNPs provide a route forward towards correcting DNA mutations responsible for genetic disease.