Pre-mRNA splicing is the most critical process in gene expression regulation across eukaryotic species. This reaction is carried out by the spliceosome, a large, dynamic, and well-organized ribonucleoprotein complex. The spliceosome is composed of five major small nuclear RNAs and an excessive number of associated protein factors. Many protein splicing factors bind and release during splicing to assist the assembly and the modulation of many RNA structures and proteins within the spliceosome. CWC25 is a splicing protein factor that functions in modulating the conformational structure of the spliceosome at the first transesterification reaction. CWC25 binds with its N-terminus to the major groove of the catalytic spliceosome triggering the spliceosome activity. Here, we employed CRISPR/Cas9 genome engineering system for targeted mutagenesis to generate CWC25 functional knock-out mutants to understand its molecular function, contribution to splicing regulation and implication in fine-tuning responses to abiotic stress in rice. Our genotyping analysis of the OsCWC25 locus revealed the presence of two mono-allelic and 18 bi-allelic mutant lines. Phenotypic analysis of these mutants, including germination and root inhibition assays, showed that the cwc25 mutants are oversensitive to abiotic stresses such as ABA and salinity. Our data demonstrate that CWC25 plays an important role in regulating plant responses to abiotic stresses.

At the post-transcriptional level, the splicing of the pre-mRNA plays a vital role in cell fate determination and respond to biotic and abiotic stresses. Through alternative splicing, mRNAs variants can be produced from a single gene. SF3B is a heptameric protein complex that is essential for pre-mRNA splicing. It contains seven subunits: SF3b155, SF3b130, SF3b145, SF3b49, SF3b14b, P14/SF3b14a and SF3b10 and they play an important role in BS (branch point sequence) recognition. P14/SF3b14a interacts with the branch point Adenosine (BPA), directing the binding of U2 complex. Several studies performed on the mutations of SF3b complex as it is associated with many diseases. Further studies are needed to deeply analyze the molecular function of P14/SF3b14a in plant growth and development. CRISPR/Cas9 system employed in gene editing among eukaryotes. The capability of the system is not only limited to the scope of bioengineering but also for functional studies of genes. CRISPR/Cas9 system assists in revealing the function of genes and the genetic networks through establishing a functional knockout and can help in understanding the molecular basis behind these processes. Here, we report the successful targeted mutagenesis of SF3b14a/p14 gene in Oryza sativa and the recovery of homozygous and heterozygous mutants. Phenotypic analyses have shown that SF3b14a/p14 is hypersensitive to abiotic stresses compared to the wild type plants. Further physiological and molecular studies are needed to reveal the role of p14 during plant growth and development, and responses to abiotic stresses.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22707号 / 農博第2423号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5300(農学部図書室) / 京都大学大学院農学研究科地域環境科学専攻 / (主査)教授 本田 与一, 教授 田中 千尋, 教授 吉村 剛 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Human immunodeficiency virus still remains the leading cause of death globally including women of child-bearing age. The rate of AIDS-related death has significantly declined since the introduction of antiretroviral treatment and other non-medical interventions such as the distribution and use of condoms. The introduction of antiretroviral treatment has however led to insulin resistance amongst users. Clustered regularly interspaced short palindromic repeats (CRISPR) CRISPR-associated nuclease 9 (Cas) has been used to knockout NFκB to understand the pathway at which antiretroviral treatment causes insulin resistance. Heteroduplex mobility assay has shown that CRISPR-Cas9 knock out the gene of interest. These results have played a foundation in understanding how CRISPR-Cas9 can be integrated and utilized in medical research. / Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020. / National Research Foundation (NRF) / Chemical Pathology / MSc (Chemical Pathology) / Restricted

Zebrafish is a frequently used model organism with an array of transgenic lines that have been used indevelopmental and physiological studies. We aim to generate novel transgenic zebrafish reporter lines to study subpopulations of spinal neurons in vivo. The gene editing system called CRISPR/Cas9 system was used to knock in reporter genes such as green fluorescent protein (GFP) or Gal4 transcription factor, to generate transgenic fish lines. Zebrafish embryos were injected with gRNAs targeting gabrb1 or nr4a2a and GFP or Gal4 plasmid, respectively. F0 larvae were screened, positive fish were raised until sexual maturity, and founders characterized to verify germline insertion. Three founders were found for gabrb1 and the location and the direction of the insert verified. The GFP expression was studied during development and differential expression patterns were identified whereas all founders had expression in brain and spinal cord. In parallel, positive fish from the Gal4 injections were raised and will be screened. Immunohistochemistry was performed to check if nr4a2a is expressed in the same cells as known neuronal markers. However, no co-localization was detected. The three gabrb1 founders identified in this study highlight the challenges into creating stable transgenic lines recapitulating true expression of the gene of interest. Sequencing, in-situ hybridization and immunohistochemistry should be performed to verify the line. A possible reason for the varying expression may be that through the knock-in we may interfere with regions regulating gene. The nr4a2a-Gal4 line will be used to perform functional studies. Those experiments will be performed using reporter genes, such as opsins or GCaMP, controlled by Upstream Activation Sequence (UAS). These transgenic lines will provide important insights regarding neuronal subpopulations that express gabrb1 and nr4a2a to unravelhow the locomotor network is formed.

The development of molecular tools that enable precise manipulation and control of biological systems would allow for a broader understanding of cellular functions and applications in biotechnology, synthetic biology, and therapeutic research. The discovery of CRISPR/Cas systems and the understanding and repurposing of their mechanisms have revolutionized the field of molecular biology. Here, I identified and characterized novel CRISPR/Cas systems and applied them for different in vivo and in vitro applications. In this work, I interrogated various Cas13 effector proteins and identified the most efficient Cas13 effector (CasRx) for in planta applications. I adapted CasRx to engineer plant immunity against different plant RNA viruses. CasRx showed robust activity and specificity against RNA viruses, demonstrating its suitability for studying key questions relating to virus biology. To expand the Cas13 toolbox and enable new applications, I performed a homology search of Cas13 enzymes in prokaryotic genomes and metagenomes, and identified previously uncharacterized, novel CRISPR/Cas13 effector proteins. I first identified and functionally characterized a small size, miniature Cas13 effector (named here as mCas13) and combined it with isothermal amplification to develop a simple and sensitive CRISPR-based SARS-CoV-2 diagnostic platform. In addition, I discovered and biochemically characterized the first known thermostable Cas13 proteins and showed that these thermostable proteins are phylogenetically related. I harnessed the unique features of these thermostable enzymes to develop the first one-pot, RT-LAMP coupled Cas13-based nucleic acid detection assay, which was utilized for highly sensitive, specific, and easily programmable detection of SARS-CoV-2 and other viruses. Lastly, I utilized CRISPR/Cas12a to develop a detection assay of plant ssDNA geminiviruses with easy-to-interpret visual readouts, making it suitable for point-of-use applications. In addition, I leveraged the self vs. non-self-discrimination and pre-crRNA processing capabilities of CRISPR/Cas12a, with the allosteric transcription factors (aTFs)- regulated expression of CRISPR array to engineer a field-deployable small molecule detection platform. I demonstrated the ability of the developed platform to detect different tetracycline antibiotics with high sensitivity and specificity. In conclusion, my work demonstrates that the discovery and characterization of programmable nucleic acid targeting systems could enable their utility for biotechnological innovations, including technologies for inhibition of viral replication and diagnostics.

The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.

Thesis advisor: Katie Rapier / Through the years, people began to breed their dogs and cats with the intention for the pets to be useful in a certain skill. However, that has shifted to focus on their looks rather than their skills thanks to kennel clubs imposing standards on each breed. This has led irresponsible breeding practices to occur which in turn caused breeds to evolve negatively as breeds began to suffer from preventable genetic disorders and negative physical changes. Genetically manipulating soon shifted from pets to humans with PGD/IVF and CRISPR-Cas9. At first, there was a focus to use these methods to help cure and prevent genetic disorders. That has since shifted to people wanting to create the perfect child. In this thesis I will argue that designer breeds help lead the way to designer babies, and that any genetic manipulation to embryos should only be done if a medical reason is present. / Thesis (BA) — Boston College, 2020. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Departmental Honors. / Discipline: Philosophy.

Gene editing with the CRISPR/Cas9 system is one of the options that sets a new trend in the development of gene therapy. The most commonly used delivery of DNA into the cells are via viruses. Nevertheless, they are often unable to take CRISPR/Cas9 system, which can be bigger than several kb. Nanoparticles (NPs), as non-viral transporters, seem to be a good alternative delivery system. For this work magnetic Fe3O4 NPs (MNPs) were selected, because of their excellent properties such as multifunctionality, biocompatibility, easy degradation and simple synthesis. The aim of this work was to synthesise MNPs and a complex of MNPs coated with PEI/CRISPR-Cas9 plasmid and to characterize them by physicochemical methods. The created complex MNPs/PEI/CRISPR-Cas9 was defined by exact parameters that are suitable for possible cell uptake. The hypothesis of stabilization of the MNPs/CRISPR-Cas9 plasmid complex by polyethylenimine (PEI), which can also protect plasmid DNA against restriction endonucleases, was verified. Next a stable modified cell line HEK293-TLR3, designed to evaluate the efficacy of double strand break (DSB) repair by nonhomologous end joining (NHEJ) or homologous recombination (HR) was, transfected with the synthesised MNPs/PEI/CRISPR-Cas9 complex. The results indicate a 25% transfection...

The question of delivery has become a critical part of therapeutic research and development. While nanoparticle formulations are now used in a variety of FDA-approved therapies, these focus on simple oral formulations or systemic lipid nanoparticle administration; therefore, significant research is ongoing in the development of specialized delivery systems for more complex cargos or specific targeting[1]. For therapeutics that have high potential for toxicity, like chemotherapeutics, delivery systems need to transport disproportionately into target organs and further target cells. This is more so true for therapeutics that require delivery to a specific cellular compartment to have significant efficacy, like in the case of gene editing in the nucleus or mitochondria-targeted peptides. To contribute to the development of efficient carriers for gene and drug therapy, this work aims to explore the potential of liposome-coated mesoporous silica nanoparticles and engineering methods that provide functionality to defeat barriers to efficient therapeutic development. Recent advances in CRISPR/Cas9 technology present an attractive toolset to study genes for the development of novel therapeutics. Since traditional delivery methods for gene therapies relied heavily on viruses— requiring biosafety level clearance and eliciting immunogenicity concerns, as well as limitations in multiplex gene editing capabilities in single viral vehicles— recently, nanoparticles have become an attractive nonviral alternative for gene therapy research and development. While lipid-based nanoparticles have been at the forefront of siRNA and mRNA therapies, we looked at increasing the loading capabilitiesof a liposome by using a mesoporous silica nanoparticle (MSN) core. The MSN provides efficient electrostatic loading of the relatively large and non-uniformly charged CRISPR/Cas9 protein and guide RNA ribonucleoprotein complex (RNP) as well as more charge-dense plasmids, while a liposome coating offers the PEGylation and targeting capabilities necessary for selective uptake and in vivo application. After demonstrating gene-editing efficiencies above 20% for both plasmid and RNP modalities of CRISPR/Cas9, we tested its application in multiplex gene editing for the study lipid metabolism pathways. To demonstrate the maintained efficiency of this system, this liposome-coated MSN (lipoMSN) platform was used to deliver a combination of RNPs targeting three genes involved in lipid metabolism in the liver. These genes, Pcsk9, Apoc3, and Angptl3, are derived from research demonstrating that populations with loss-of-function mutations in any one of these genes garner improved cardiovascular health, characterized by lowered blood cholesterol and triglycerides. The lipoMSN system demonstrated that it maintained significant gene editing, above 25% efficiency, at a specific gene target despite reduced dosage of target-specific RNP due to the combination of other target RNP. By leveraging this system to deliver various combinations of targeting RNPs in the same nanoparticle and therefore ensuring a higher probability that any given cell is edited at all targets, synergistic effects on lipid metabolism can be observed in vitro and in vivo. These effects, such as an approximately 50% decrease in serum LDL-cholesterol 4-weeks after treatment with pcsk9 and angptl3- targeted RNPs, have not been observed in previous studies. Continued work with this lipoMSN platform is ongoing, with projects leveraging the system for multiplex gene disruption as well as endosomal delivery of peptide and chemical drugs. We are currently leveraging the comparatively low spread of lipoMSN to provide gene disruption of adra1a, adra1b, and adra1d in the discrete area of the thalamus. Further, this system is also well suited for other CRISPR/Cas9 elements, such as deactivated Cas9 (dCas9) fused to epigenetic modifiers, enhancers and repressors. To demonstrate another facet of the lipoMSN delivery platform, we adjusted the formulation for the specific delivery to endosomal compartments of nociceptive neurons. Previous work by our collaborators at the Bunnett Lab provided evidence that signaling in the endosomal compartment is partially responsible for both pain propagation and amelioration. In order to provide highly targeted treatment to reduce the off-target effects linked with opioid-based pain treatment, we wanted to leverage the resonance of these nanoparticles in the endosomes following endocytosis and enhance the drug delivery to the endosomal compartments propagating nociceptive signaling. We did this by first integrating a targeting ligand in the form of a DADLE-peptide, to the outside of the liposomes, providing a 20-40% increase in uptake for delta-opioid receptor (DOR)-expressing cell types. Further, we used an oxidation- and pH-sensitive MSN core to provide increased drug release to the endosomal environment, which is naturally oxidizing and at a lower pH of approximately 5.2. This resulted in an increased therapeutic effect when compared to naked peptide drug in a mouse model of neurogenic pain. We are also looking at leveraging this system for other endosomal signaling pathways and applying our lipoMSN platform to cargos such as glucagon-like peptide-1 (GLP-1) receptor agonists for diabetes and US28 receptor antagonists for reduction of proliferative signaling in cancers. Collectively, these projects provide insight on how to design delivery vehicles for specific gene and drug delivery. This lipoMSN system has the potential to be a versatile platform for the development of combinatorial gene therapeutics in liver-related disease. Further, this platform may inform research and development in the next generation of endosomally-targeted therapeutics for increased efficacy and reduced off-target or side effects.