Programmable site-specific endonucleases are useful tools for genome editing and may lead to novel therapeutics to treat genetic diseases. TALENs can be designed to cleave chosen DNA sequences. To better understand TALEN specificity and engineer TALENs with improved specificity, we profiled 30 unique TALENs with varying target sites, array length, and domain sequences for their ability to cleave any of 1012 potential off-target DNA sequences using in vitro selection and high-throughput sequencing. Computational analysis of the selection results predicted 76 off-target substrates in the human genome, 16 of which were accessible and modified by TALENs in human cells. The results collectively suggest that (i) TALE repeats bind DNA relatively independently; (ii) longer TALENs are more tolerant of mismatches, yet are more specific in a genomic context; and (iii) excessive DNA-binding energy can lead to reduced TALEN specificity in cells. We engineered a TALEN variant, Q3, that exhibits equal on-target cleavage activity but 10-fold lower average off-target activity in human cells. Our results demonstrate that identifying and mutating residues that contribute to non-specific DNA-binding can yield genome engineering agents with improved DNA specificities.

The ability to precisely manipulate the genome in a targeted manner is fundamental to driving both basic science research and development of medical therapeutics. Until recently, this has been primarily achieved through coupling of a nuclease domain with customizable protein modules that recognize DNA in a sequence-specific manner such as zinc finger or transcription activator-like effector domains. Though these approaches have allowed unprecedented precision in manipulating the genome, in practice they have been limited by the reproducibility, predictability, and specificity of targeted cleavage, all of which are partially attributable to the nature of protein-mediated DNA sequence recognition. It has been recently shown that the microbial CRISPR-Cas system can be adapted for eukaryotic genome editing. Cas9, an RNA-guided DNA endonuclease, is directed by a 20-nt guide sequence via Watson-Crick base-pairing to its genomic target. Cas9 subsequently induces a double-stranded DNA break that results in targeted gene disruption through non-homologous end-joining repair or gene replacement via homologous recombination. Finally, the RNA guide and protein nuclease dual component system allows simultaneous delivery of multiple guide RNAs (sgRNA) to achieve multiplex genome editing with ease and efficiency. The potential effects of off-target genomic modification represent a significant caveat to genome editing approaches in both research and therapeutic applications. Prior work from our lab and others has shown that Cas9 can tolerate some degree of mismatch with the guide RNA to target DNA base pairing. To increase substrate specificity, we devised a technique that uses a Cas9 nickase mutant with appropriately paired guide RNAs to efficiently inducing double-stranded breaks via simultaneous nicks on both strands of target DNA. As single-stranded nicks are repaired with high fidelity, targeted genome modification only occurs when the two opposite-strand nicks are closely spaced. This double nickase approach allows for marked reduction of off-target genome modification while maintaining robust on-target cleavage efficiency, making a significant step towards addressing one of the primary concerns regarding the use of genome editing technologies. The ability to multiplex genome engineering by simply co-delivering multiple sgRNAs is a versatile property unique to the CRISPR-Cas system. While co-transfection of multiple guides is readily feasible in tissue culture, many in vivo and therapeutic applications would benefit from a compact, single vector system that would allow robust and reproducible multiplex editing. To achieve this, we first generated and functionally validated alternate sgRNA architectures to characterize the structure-function relationship of the Cas9 protein with the sgRNA in DNA recognition and cleavage. We then applied this knowledge towards the development and optimization of a tandem synthetic guide RNA (tsgRNA) scaffold that allows for a single promoter to drive expression of a single RNA transcript encoding two sgRNAs, which are subsequently processed into individual active sgRNAs.

Parkinson’s disease is a highly prevalent multifactorial neurodegenerative disorder caused by a complex cascade of interactions between various genetic and environmental factors. Due to this, the majority of cases are termed idiopathic. However, about 10% of PD cases are due to defined genetic factors. Interestingly, both idiopathic and familial cases of PD share mitochondrial dysfunction as a central component in the pathology of the disease. The mitochondrial protease, presenilin-associated rhomboid-like (PARL), is one such Parkinson's disease-linked gene, and is associated with diverse processes including mitochondrial dynamics, active inhibition of unnecessary apoptosis and mitophagy in Drosophila and yeast. Here, I investigated the role of the two zebrafish parl paralogs, parla and parlb, through stable CRISPR-Cas9 mediated mutagenesis. I injected wild type embryos with sgRNAs targeting parla and parlb loci, successfully producing indel mutations in parlb and multi-exon deletions in parla at mutation efficiencies of 74% and 40%, respectively. Through whole mount in situ hybridization experiments against th1, I saw no change in dopaminergic (DA) neuron development displayed by parlb mutants compared to wild types. Injection of parla splice blocking morpholinos into parlb mutants indicates that proper DA neuron development may depend principally on Parla function and loss of both Parla and Parlb function increases larval mortality. These results suggest a negative epistatic relationship between the parl paralogs as seen by the more severe phenotype observed in the loss of both Parla and Parlb function compared to the individual effects.

CRISPR (clustered regularly interspaced short palindromic repeats)-guided nucleases such as Cas9 remain atop the most exciting biological systems in gene editing and therapeutic potential. The modern adaptation of the Streptococcus pyogenes (Spy) Cas9 machinery for directed DNA editing relies on two major components; a Cas9 protein, and an RNA guide. This study aims at modification of the guide RNA to probe structural requirements and modification tolerance while studying the biochemical properties of the ribonucleoprotein complex. We find that the SpyCas9-RNP is not only capable of heavy substitutions and modifications in the guide, but specific properties can be tuned by careful and directed modifications. DNA substitutions in the 3’ end of the guide RNA improves cleavage activity and efficiency in vitro. Various RNA chemistries such as 2’-fluoro and other RNA mimics maintain biochemical activity but can be used to improve stability against nucleases. Additionally, tethering of the tracrRNA and crRNA together into a sgRNA was found to affect the fundamental properties of the Cas9 protein, improving activity, but reducing target binding affinity and cleavage rate. Directed modification of the guide RNA can be used to exploit certain biochemical properties for effective therapeutic applications.

The nonapeptide family of hormones is deeply conserved in evolution. In teleost fishes, as in all vertebrates, two nonapeptide families exist. These are vasotocin (avp) and oxytocin (oxt). While vasotocin has been shown to regulate individual aspects of reproductive physiology in several teleost species, an integrative assessment of its role on male and female reproduction is currently lacking even in widely used fish models, such as the zebrafish (Danio rerio). Taking advantage of the genetic tractability of the zebrafish, and its emerging status as model to study reproductive physiology, I generated avp -/- mutants using a CRISPR/Cas9 based approach to determine reproductive consequences in female and male zebrafish. Following the identification of a female-specific reproductive phenotype which manifests as a reduction in oocyte release and decreased quivering behaviour, I investigated the potential mechanistic basis at the level of the gonad. In avp -/- ovaries, significantly fewer eggs were present compared to WT fishes. When comparing the distribution of oocyte maturation stages, a significantly lower percentage of stage I and higher percentage of stage V oocytes was present in avp-/- ovaries. The altered distribution in oocyte maturation stages coincided with significant decreases in ovarian transcript abundance of nanos2, a germ-cell specific marker suggesting a possible role for Avp in germ-cell maintenance. Additionally, I observed a decrease in the ovarian concentration of the prostaglandin PGF2, which coincided with a reduction in ovarian transcript abundance of pla2g4ab, a paralogue of the phospholipase A2 involved in mobilizing arachidonic acid, a precursor of PGF2,. Together, these finding suggests a role for Avp in PGF2 -mediated ovulation. Because Avp has pleiotropic effects and may thus affect female reproductive physiology indirectly, we assessed somatic growth, a key regulator of sexual maturation in zebrafish, as well as aspects of the endocrine stress axis known to affect oocyte growth in avp -/- mutants. While avp -/- mutants did not exhibit differences in somatic growth up to sexual maturation or GSI, mutants exhibited hypercortisolism. While other zebrafish knock-out mutants exhibiting persistent hypercortisolism do not share the observed reproductive phenotype, future studies investigating potential contributions of pleiotropic Avp effects are nevertheless warranted. Overall, I demonstrate that avp, while not essential, affects female reproductive success, at least iii in part by regulating oocyte maturation. This finding is in line with the recent findings from other vertebrate and invertebrate species, suggesting an evolutionarily ancient role in these processes. It is anticipated that such novel insights into the regulation of female oocyte maturation have in addition to increasing our understanding of female reproduction, translational potential for captive breeding (aquaculture, species conservation) and ecotoxicology (insight into mode of action of specific EDCs).

Fusarium graminearum is responsible for causing Fusarium head blight in cereals and maize imposing a significant impact in Canadian agriculture. While a handful of secondary metabolites produced by F. graminearum are recognized as contributors to disease virulence, the functions of numerous molecular products arising from biosynthetic gene clusters expressed during infection remain undiscovered. Presented here are the results of CRISPR-Cas9 mediated gene-deletion experiments disrupting core biosynthetic genes from four biosynthetic gene clusters with reported in-planta transcription: C08, C16, C13 and C70. Both wheat head infection assays and coleoptile infection assays were used to evaluate the pathology phenotypes of transformant strains illustrating potential links between C16 and pathogenicity. Culture medium screening experiments using transformant strains were profiled by UHPLC-HRMS and targeted MS2 experiments to confirm the associated secondary metabolite products and attempt to identify unknown secondary metabolites of the biosynthetic gene clusters. While C08 secondary metabolite remained elusive, confirmation of C16 secondary metabolites led to hypotheses regarding their potential connections to the inhibition of plant immune response and untargeted secondary metabolite profiling of the C13/C70 transformant strains suggests that this BGC may have significant implications for global secondary metabolite production.

Los sistemas de defensa de procariotas denominados CRISPR–Cas dan lugar a la formación de un complejo nucleoproteico compuesto por proteínas Cas y un ARN guía derivado de las regiones CRISPR que se encarga de interferir en la propagación de elementos genéticos móviles a través de la degradación mediada por nucleasas Cas de secuencias foráneas que coinciden con los ARN guía. La fácil reprogramación de la guía ha propiciado el desarrollo de un arsenal de herramientas para la modificación y manipulación de ácidos nucleicos, cuyas aplicaciones superan con creces la utilización implementada inicialmente para la proteína Cas9 de Streptococcus pyogenes como editor del ADN. En un intento de paliar las limitaciones encontradas en el uso de la Cas9 de S. pyogenes, que sigue siendo la herramienta CRISPR más empleada, el repertorio de nucleasas Cas disponibles ha aumentado considerablemente en los últimos años, en particular las pertenecientes a la clase 2. La actividad de corte inespecífico fuera de la diana, el requerimiento de un motivo concreto adyacente a la diana y su gran tamaño, que limita su administración celular, han motivado la búsqueda de nuevas nucleasas en datos genómicos y metagenómicos, así como la generación de variantes derivadas de proteínas nativas mediante evolución dirigida o ingeniería de proteínas. En la presente tesis, llevamos a cabo una búsqueda de sistemas CRISPR–Cas de clase 2 a partir de 10 metagenomas provenientes de tres ambientes previamente inexplorados en este sentido, dando como resultado la identificación de 107 sistemas pertenecientes a alguno de los subtipos descritos hasta la fecha. El análisis de las características de las proteínas Cas encontradas y su relación evolutiva con otras ortólogas empleadas en edición genética nos llevó a la selección de 9 candidatas para su caracterización funcional. Entre todas ellas, la proteína EH6Cas9 mostró actividad nucleasa sobre secuencias diana de ADN en E. coli, adyacentes al motivo 5’-NGG-3’. Con el fin de desarrollar una nueva herramienta de biología molecular, la simplificación del sistema nos llevó a diseñar un ARN guía sintético compatible con EH6Cas9. La caracterización bioquímica de la herramienta demostró que EH6Cas9 es una ADNasa metal dependiente, programable mediante guías de ARN, que muestra su máxima actividad frente a dianas adyacentes a la secuencia consenso 5’-NGGDT-3’ a temperaturas entre 25 °C y 37 °C. Finalmente, EH6Cas9 también demostró su aplicabilidad como herramienta de edición genética. En E. coli, EH6Cas9 mostró una gran eficacia como estrategia para la selección positiva de mutantes, mientras que en células de ratón indujo la formación de inserciones y deleciones en una secuencia diana, presentando características distintivas respecto a la Cas9 de S. pyogenes que la hacen especialmente indicada para aplicaciones de edición genética en eucariotas en las que se requiera una actividad nucleasa moderada y más restringida.

This thesis proposal shows the efficiency of different methods of Myostatin inhibition by using CRISPR/CAS9. With the data cataloged by this thesis, researchers will have a better understanding of what methods are better to achieve their goals. The data was collected by reading multiple scientific articles involving the usage of CRISPR/CAS9 to inhibit the Myostatin protein. The data collected from all the different studies were analyzed in the same categories. The experiment that used CRIPSR/CAS9 on in vitro specimens had a superior Myostatin inhibition overall, therefore presenting higher muscle mass. The method using CRISPR/CAS9 to inhibit the Myostatin in vivo and in vitro depends on what the researcher is trying to accomplish. By reading this thesis researchers can understand that if they choose to use an in vitro treatment the results would be way more severe than using an in vivo application treatment.

Giardia intestinalis is a unicellular protozoan parasite, causing giardiasis – a gastro-intestinal disease of variable outcome and severity, in a broad range of mammalian species. The parasite has a comparatively simple life cycle with two stages, proliferative trophozoites and infectious cysts, as well as a reduced set of eukaryotic-specific organelles. Its metabolic pathways are simplified if compared to non-parasitic organisms. Giardia compensates for this apparent simplicity with unique inventions and complex regulation of metabolic processes. Transition from a fragile trophozoite to a protected, compact cyst is called encystation. This process starts upon changes in the growing conditions: cholesterol deprivation and elevated pH, and leads to changes in membrane lipids, elevated cAMP, and induction of encystation-specific gene expression, starting with activation of Myb-like protein expression. Within hours postencystation induction, cyst wall components (GalNAc and CWPs) are produced and transported to the cell membrane, flagella and adhesive disc are disassembled and stored in cytoplasm, followed by DNA replication and diplomixis. One of the encystation-specific upregulated genes in Giardia (assemblage A, isolate WB) is GL50803_1470 (termed ORF1470). Its predicted protein product has an Alba_2 domain, binding nucleic acids, presumably DNA. To study its function in G. intestinalis, we created knockout mutants, using CRISPR/Cas9 technique, Cas9- HA cell line and pGdelp-BbsI-B00826, with integrated pac- and gRNA cassettes. Transfected Giardia Cas9-HA cells were PCR verified for the complete knockout of the gene, encysted, and effect of ORF1470 was studied using cyst counting, morphometric analysis, cell imagining and Western blot for detection of CWPs. We have found minor phenotypical differences between the parental strain Cas9-HA and wild-type WB and ORF1470 deficient cells. Future plans are further experiments with obtained ΔORF1470 strains including further KO verification, visualization of ORF1470 product during encystation and determination of its binding site in the genome using ChIP-seq technology.

Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Thus, methods to accelerate genetic improvement to improve woody biomass yield, quality and optimal growth on marginal lands are of considerable interest. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, co-orthologs of Arabidopsis SECONDARY CELL WALL NAC DOMAIN2 (SND2), which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, members of the TERMINAL FLOWER1 (TFL1)/CENTRORADIALIS (CEN) gene family that act as flowering repressors in Arabidopsis and many plants. I studied INRA 717-1B (P. tremula x P. alba) transgenics with an artificial microRNA (AmiRNA) downregulating PopNAC154 and PopNAC156 (AmiSND2 trees). In a field trial, AmiSND2 trees showed higher mean height and diameter than wild-type (WT). We also observed that AmiSND2 transgenics showed delayed leaf senescence and leaf drop. After conducting controlled environment studies with AmiSND2 trees, I was able to confirm that downregulation of PopNAC154 and PopNAC156 genes does not alter the short daylength-induced bud set and growth cessation but it delays the low temperature induced leaf senescence and leaf drop. Further I was able to show that down regulation of the PopNAC154 and PopNAC156 genes resulted in significantly higher mean plant heights and delayed bud set compared to the WT plants under low soil nutrient conditions. Wood chemistry data analysis of field grown AmiSND2 trees showed that they have a significantly higher cellulose content a lower lignin content compared to that of the WT. Thus, these results show that downregulating the PopNAC154 and PopNAC156 genes has the potential to increase biomass yield and quality. In a previous study, simultaneous downregulation of PopCEN1 and PopCEN2 genes using RNA interference (RNAi) method caused poplar trees to flower only after two years of growth in the field. I used CRISPR/CAS9 method to knock-out each paralog individually as well as the related gene, PopBFT. The popcen1 mutant trees developed flowers even under in vitro conditions, but popcen2 mutants did not show an obvious phenotype. popbft mutant trees also did not show an obvious phenotype under standard growing conditions. However, when soil nutrient availability was allowed to deplete, the popbft mutants showed lower mean plant height compared to the WT and also showed lower root length and root volume under low Nitrogen conditions in an in vitro assay compared to the WT. These results prove that PopCEN1 gene is directly involved in repressing flowering in poplar and allele-specific mutation should be tested as an approach to accelerate breeding. PopCEN2 and PopBFT might not have a role in regulation of flowering time, and though additional studies are needed, PopBFT appears to have a role in regulating growth in response to nutrient availability. / Master of Science / Poplar (Populus spp. and hybrids) are used for pulp, paper and solid wood products. Furthermore, poplar is being developed as a dedicated biomass crop for biofuels and biomaterials. Genes control the woody biomass yield, and quality among all the other characteristics, thus, methods to accelerate genetic improvement to improve these characteristics is paramount. One approach is to identify genes that could be manipulated through breeding or biotechnology to achieve these goals. I studied two sets of candidate genes for improving biomass, growth and manipulating flowering time. First, I studied the functions of PopNAC154 and PopNAC156, genes which are putative regulators of wood cell wall synthesis, the source of lignocellulosic biomass. Second, I studied PopCEN1, PopCEN2, and PopBFT, genes that act as flowering repressors in Arabidopsis and many plants. I studied a genetically modified poplar, where PopNAC154 and PopNAC156 genes’ function were reduced. After subjecting these plants to shorter daylength periods and cold temperatures in controlled conditions I was able to show that decreased activity of the PopNAC154 and PopNAC156 genes causes the trees to slowdown the leaf senescence and retain their leaves under low temperature conditions compared to the genetically unaltered wildtype (WT) poplar. I was also able to show that decreased activity of the PopNAC154 and PopNAC156 genes allow the poplar plants to grow better than the WT plants under low soil nutrient conditions. I used gene editing to switch off PopCEN1, PopCEN2, and PopBFT genes. When PopCEN1 gene was switched off individually the young mutant trees flowered whereas normally trees take six to eight years to flower. However, when the PopCEN2, and PopBFT genes were switched off individually the resulting mutant plants did not show any signs of flowering. Hence, I was able to show that PopCEN1 gene is directly involved in repressing the flowering in poplar plants. In conclusion, my work identified PopNAC154 and PopNAC156 as candidate genes for manipulation by breeding or biotechnology to increase wood yield, and suggested ways to induce flowering to accelerate breeding through manipulation of PopCEN1.