Browsing by Subject "CRISPR"

Mycobacterial infection leads to the formation of characteristic immune cell aggregates called granulomas. In humans and animal models, tuberculous granuloma formation is accompanied by dramatic remodeling of host vasculature which ultimately benefits the infecting mycobacteria, suggesting the bacteria may actively drive this host process. First, we sought to identify bacterial factors that promote granuloma vascularization. Using Mycobacterium marinum transposon mutants in a zebrafish infection model, we revealed the enzyme Proximal Cyclopropane Synthase of alpha-Mycolates (PcaA) as an important bacterial determinant of host angiogenesis. We found that PcaA-modified trehalose dimycolate, an abundant glycolipid in the mycobacterial cell wall, drives activation of host VEGF signaling and subsequent granuloma vascularization. To facilitate our continuing investigation of granuloma dynamics, we next sought to expand and improve upon the transgenic tools for studying macrophages in the zebrafish model. I describe two such tools: i) the macrophage-specific zebrafish mfap4 promoter, which allows long-term in vivo visualization and manipulation of macrophages during mycobacterial infection, and ii) the first zebrafish transgenic line with constitutive, ubiquitous Cas9 expression, as well as a transgene design capable of generating sgRNAs using macrophage-specific promoters. These tools allow CRISPR/Cas9 gene editing in vivo in the zebrafish in a macrophage-restricted manner.

The eukaryotic epigenome has an instrumental role in determining and maintaining cell identity and function. Epigenetic components such as DNA methylation, histone tail modifications, chromatin accessibility, and DNA architecture are tightly correlated to central cellular processes, while their dysregulation manifests in aberrant gene expression and disease. The ability to specifically edit the epigenome holds the promise of enhancing understanding how epigenetic modifications function and enabling manipulation of cell phenotype for scientific or therapeutic purposes. Genome targeting technologies, such as the CRISPR/Cas9 system, have successfully been harnessed to create epigenome editing tools to alter gene expression. Prominently, two leading CRISPR-based technologies, CRISPRa and CRISPRi, were shown to be highly specific and effective in controlling gene transcription levels. These tools, however, often lead to formation of complexes that affect a multitude of endogenous factors, thus mitigating our ability to elucidate the role of individual epigenetic marks. Moreover, changes in epigenetic marks are associated with numerous health conditions, therefore the development of tools that can modify specific marks may help in creating disease models, or the restoration of a “healthy” epigenome. We first created a suite of CRISPR-based epigenome modifiers (CRISPR-GEMs) that were aimed to catalyze the removal or addition of specific histone tail marks. Next, we tested a few promising CRISPR-GEMs on multiple target genes to characterize their effect on gene expression and chromatin marks. Furthermore, we utilized these tools to deepen our insights into the relationship of individual histone marks and gene expression in different contexts and to better our understanding of the kinetics and dynamics of several of these novel tools alongside existing ones. Additionally, we decided to use the CRISPRa platform to explore senescence, a cellular process that is at the epicenter of aging and has been shown to play a key role in various age-related diseases. Using the CRISPRa platform in an inducible-senescence cell model, we found and validated multiple transcription factors (TFs) that regulate senescence-associated growth arrest (SAGA). Lastly, we characterized genetic pathways that are pivotal to successful inhibition of SAGA, thereby demonstrating a new application of epigenome editing in a senescence model that enhanced our understanding of the pathways that govern SAGA.

The differentiation of human induced pluripotent stem cells (hiPSCs) to prescribed cell fates enables the engineering of patient-specific tissue types, such as hyaline cartilage, for applications in regenerative medicine, disease modeling, and drug screening. In many cases, however, these differentiation approaches are poorly controlled and generate heterogeneous cell populations. In this dissertation, we demonstrate robust cartilaginous matrix production in multiple hiPSC lines using a robust and reproducible differentiation protocol. To purify chondroprogenitors produced by this protocol, we engineered a COL2A1-GFP knock-in reporter hiPSC line by CRISPR-Cas9 genome editing. Purified chondroprogenitors demonstrated an improved chondrogenic capacity compared to unselected populations, improved matrix homogeneity, and reduced variability between tissues. We next demonstrated the ability of the system to serve as a high-throughput system for arthritis disease modeling using cytokine stimuli. Finally, we used this platform to screen for transcription factors whose activation might be involved in chondrogenic lineage specification of hiPSCs. Taken together, these studies describe the generation of a high-throughput system for chondrogenesis and its application for screens and arthritis disease modeling. Future applications of this platform may be useful for identifying pathways regulating cartilage regeneration and novel therapeutics for arthritis.

Genome-wide association studies have identified thousands of DNA variants associated with specific phenotypes, yet it remains unknown which variants are causal. Additionally, more than 90 percent of common variants occur in noncoding genomic regions, further complicating efforts to predict their function. Large consortia efforts have leveraged functional genomics assays to characterize the genome-wide and epigenome-wide features of noncoding regions and common candidate cis-regulatory elements. However, these predictions are largely based on DNA sequence or correlation of epigenome marks alone, and do not provide information for which genes and broader regulatory networks are controlled by these candidate cis-regulatory elements. Advancements in CRISPR/Cas9-based genome and epigenome editing tools and multiplexed screening assays have enabled systematic perturbation of candidate cis-regulatory elements. We first sought to establish principles for performing noncoding CRISPR screens. We next applied these principles to investigate a subclass of cis-regulatory elements that respond to mechanical stimuli. Finally, we combined noncoding CRISPR screening approaches with single cell transcriptome profiling to clarify the regulatory landscape in diverse cell types in the Major Histocompatibility (MHC) Locus, one of the most complex regions of the human genome. Through these efforts, we establish guidelines for noncoding CRISPR screen design, execution, and analysis, identify mechanosensitive cis-regulatory elements and their role in complex cellular processes, and reveal cell-type specific and shared regulatory mechanisms governing gene expression in the MHC locus. Collectively, these studies provide experimental and computational frameworks for future investigation of cis-regulatory element function and will enable further dissection of variant-gene-phenotype relationships.

Since being developed as a tool in 2012, CRISPR-Cas9 and other CRISPR systems have revolutionized the way we manipulate biology. CRISPR systems broadly rely on a programmable guide RNA (gRNA) to enable targeted nuclease activity. Additionally, the ability to target a protein to a specific sequence of DNA has enabled a myriad of applications, including transcriptional silencing and/or activation, single base editing and targeted transposase activity. However, there remains a gap in knowledge as to what factors influence the activity of these systems. The gRNA sequence has been shown to at least partially predict activity but the mechanism behind this is not well understood. In addition, other contextual factors such as DNA repair, target accessibility, and others may play a role. Here, we present factors that influence Cas9 activity in E. coli and other organisms. Additionally, we find that gRNA sequence influences activity in large part by determining how fast Cas9 finds the target site. Together, the work presented improves our understanding of Cas9 and could lead to better gRNA prediction algorithms and new routes to improve Cas9 on-target activity.

Targeted therapeutics are among the most promising approaches for treating diverse forms of malignancies. Indeed, as sequencing prices and technology continue to improve it will be possible to achieve a precise map of each individual cancer’s genomic lesions, providing insights into the best strategies for treatment. However, these approaches will be undermined by cancer’s ability to resist upfront target inhibition (intrinsic resistance) as well as, in cases where tumors are initially sensitive, develop resistance over the course of drug treatment (acquired resistance). The literature to date reveals a problem as complex as the tumor itself; the heterogeneity of cancer as a disease is matched by the myriad ways in which it evades treatment.

Understanding drug resistance as a whole quickly becomes a problem of scale. Not only is there cancer subtype-associated variation to consider, but also intrinsic and acquired resistance profiles can differ based on the type of inhibitor used and at what node the offending pathway is inhibited. Assigning proper treatments to account for these mechanisms adds an additional layer of complexity as the number of FDA-approved and late-stage clinical candidate molecules increases. Here, when applied appropriately, high throughput methods offer the ability to screen thousands of perturbations in parallel, quickly narrowing the search space for a phenotype of interest.

This work applies such methods to the cell-autonomous complexity of drug resistance and seeks to understand (1) mechanisms by which cancer cells evade drug treatment, (2) design concepts for the most effective combinatorial drugging strategies, and (3) how it might be possible to account for resistance-associated heterogeneity by targeting the evolutionary liabilities of resistant cells. Using a combination of open reading frame (ORF), clustered regularly spaced short palindromic repeats (CRISPR), and pharmacologic screening technologies, this work attains the resolution and throughput necessary to address 1-3 above and begins to unravel the complexity of drug resistance.

Over the past several years genome and epigenome engineering has been propelled forward by CRISPR-Cas technologies. These prokaryotic defense systems work well in mammalian cells in a manner that is remarkably robust: they are non-toxic, fold into a catalytically active state, localize to targeted cellular compartments, and act on the eukaryotic genome, which is heavily compacted in chromatin. While all these are true, CRISPR-cas nucleases did not evolve to function as highly specific genome engineering tools. Thus, the major goals of the work presented herein are to i) refine the specificity of CRISPR-Cas enzymes, ii) develop methods that facilitate genome engineering in human cells, and iii) apply these technologies toward outstanding problems in human gene regulation. With regard to the first goal, we set out to develop a method that could be easily applied to increase the specificity of diverse CRISPR systems. Adopting RNA-engineering to achieve this goal, we modulate the kinetics of DNA strand invasion to increase the specificity of Cas enzymes. Since the guide RNA is a feature that is common across all CRISPR systems, we expect that this new method to tune the activity and specificity of Cas enzymes will be broadly useful. To address the second goal, we set out to develop an experimental pipeline for the high throughput, precise modification of mammalian genomes. Specifically, we modify the C-termini of genes to include an epitope tag for the genome-wide profiling of transcription factor binding sites. We apply this method to over 30 genes, encoding a variety of transcription factors, chromatin modifying enzymes, and gene regulatory proteins. Out of the large number of genes we focus particularly on members of the AP-1 transcription factor family and nuclear receptor co-activator and co-repressor families. Using this ChIP-seq data, which profiles genome wide binding, and integrating a variety of other genomic information, including chromatin modifications, chromatin accessibility, other TF binding, and inherent regulatory activity, we investigate the dimerization preferences of AP-1 subunits, their genomic binding patterns, and the regulatory potential of theses subunits. Toward addressing the third goal, we decided to focus on the glucocorticoid receptor (GR). The dual activating and repressive function of the GR is incompletely understood, and this duality is a property of many other stimuli responsive transcriptional responses (e.g. NFKB signaling). Thus, how one transcription factor is biochemically endowed with the ability to both activate and repress gene expression is an outstanding problem in gene regulation. It is hypothesized that the GR recruits a variety of distinct protein complexes in order to mediate its diverse function. We used CRISPR based loss of function screening in order to discover new GR cofactors. Using this method, we find a number of cofactors, both canonical and novel, that regulate this response in A549 cells. Ongoing work investigates how general these cofactors are across the transcriptome and whether they provide an avenue to decouple GR’s dual function, which has been a major goal in drug development. Through these studies we have found a way to make CRISPR systems more specific, developed and applied CRISPR based method to define AP-1 binding and function, and used unbiased CRISPR based screens to discover novel regulators of the glucocorticoid drug response.

Chapter 1 broadly introduces this work, its motivations, and aims of research presented herein.

Chapter 2 provides an introduction to both genome engineering and gene regulation. Specifically, it describes the development and application of CRISPR-cas tools and details outstanding problems in gene regulation through the lens of nuclear receptors.

Chapter 3 describes the purification of Cas9 protein and its characterization biochemically. Specifically, we use AFM to determine the DNA binding properties of Cas9 in vitro.

Chapter 4 introduces a new method to modulate the specificity of CRISPR systems in human cells. Therein we show that RNA secondary structure can be applied to diverse CRISPR systems to tune their activity.

Chapter 5 details a method for the high throughput tagging of transcription factors. It specifically investigates members of the AP-1 transcription factor complex.

Chapter 6 is an investigation of the glucocorticoid receptor and its cofactors. We apply a variety of genome engineering and genomic methods to characterize known co-factors and discover new ones.

Chapter 7 is an outlook on the fields of genome-engineering and gene regulation. It describes key questions that are still unanswered and possible lines of attack to address them.

Point mutations in the active site of isocitrate dehydrogenases 1 and 2 (\textit{IDH}) occur in the majority of WHO grade II and III gliomas, resulting in a unique milieu of signaling and metabolism. IDH1/2 active site mutations confer a gain-of-function activity to the enzyme, which results in the production of the oncometabolite D-2-hydroxyglutarate (D-2HG). D-2HG accumulation in turn promotes tumor formation through competitive inhibition of $\alpha$-ketoglutarate dependent ($\alpha$-KG) enzymes. Inhibition of $\alpha$-KG-dependent enzymes, such as histone demethylases and DNA demethylases, is sufficient to induce tumor-promoting epigenetic changes, but can also impose situational constraints on cell proliferation. To develop better therapies for mutant IDH1-bearing gliomas, it is essential to determine whether the epigenetic changes induced by the mutant IDH proteins actively require the mutation after tumor formation. Furthermore, it is imperative to decode the molecular mechanisms that promote tumor cells’ fitness under IDH mutation-dependent constraints in representative models. Here, we describe and characterize CRISPR-Cas9 based isogenic cell line models using patient-derived IDH1$^{R132H/WT}$ glioma cell lines. We uncover that these models show persistent DNA hypermethylation in CpG loci of the glioma CpG island methylator phenotype even after D-2HG production has been abolished. We also report a genome wide pattern of DNA demethylation in CpG sites outside of CpG islands, which reflect the acquisition of a G-CIMP-low like state after loss of D-2HG production. Then, using these cell line tools, we performed an unbiased sub-genomic CRISPR-library screening to identify genes whose functions supported the growth of glioma cells bearing endogenous IDH1 mutations. This work thus provides new patient derived models for exploring novel therapeutic opportunities for IDH1 mutant tumors, and uncovers the extent to which IDH mutation linked hypermethylation profiles in glioma depend upon D-2HG production from the IDH mutation.

Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.

The growing complexity of scientific research demands further development of advanced gene regulation systems. For instance, the ultimate goal of tissue engineering is to develop constructs that functionally and morphologically resemble the native tissue they are expected to replace. This requires patterning of gene expression and control of cellular phenotype within the tissue engineered construct. In the field of synthetic biology, gene circuits are engineered to elucidate mechanisms of gene regulation and predict the behavior of more complex systems. Such systems require robust gene switches that can quickly turn gene expression on or off. Similarly, basic science requires precise genetic control to perturb genetic pathways or understand gene function. Additionally, gene therapy strives to replace or repair genes that are responsible for disease. The safety and efficacy of such therapies require control of when and where the delivered gene is expressed in vivo.

Unfortunately, these fields are limited by the lack of gene regulation systems that enable both robust and flexible cellular control. Most current gene regulation systems do not allow for the manipulation of gene expression that is spatially defined, temporally controlled, reversible, and repeatable. Rather, they provide incomplete control that forces the user to choose to control gene expression in either space or time, and whether the system will be reversible or irreversible.

The recent emergence of the field of optogenetics--the ability to control gene expression using light--has made it possible to regulate gene expression with spatial, temporal, and dynamic control. Light-inducible systems provide the tools necessary to overcome the limitations of other gene regulation systems, which can be slow, imprecise, or cumbersome to work with. However, emerging light-inducible systems require further optimization to increase their efficiency, reliability, and ease of use.

Initially, we engineered a light-inducible gene regulation system that combines zinc finger protein technology and the light-inducible interaction between Arabidopsis thaliana plant proteins GIGANTEA (GI) and the light oxygen voltage (LOV) domain of FKF1. Zinc finger proteins (ZFPs) can be engineered to target almost any DNA sequence through tandem assembly of individual zinc finger domains that recognize a specific three base-pair DNA sequence. Fusion of three different ZFPs to GI (GI-ZFP) successfully targeted the fusion protein to the specific DNA target sequence of the ZFP. Due to the interaction between GI and LOV, co-expression of GI-ZFP with a fusion protein consisting of LOV fused to three copies of the VP16 transactivation domain (LOV-VP16) enabled blue-light dependent recruitment of LOV-VP16 to the ZFP target sequence. We showed that placement of three to nine copies of a ZFP target sequence upstream of a luciferase or eGFP transgene enabled expression of the transgene in response to blue-light. Gene activation was both reversible and tunable based on duration of light exposure, illumination intensity, and the number of ZFP binding sites upstream of the transgene. Gene expression could also be spatially patterned by illuminating the cell culture through photomasks containing various patterns.

Although this system was useful for controlling the expression of a transgene, for many applications it is useful to control the expression of a gene in its natural chromosomal position. Therefore we capitalized on recent advances in programmed gene activation to engineer an optogenetic tool that could easily be targeted to new, endogenous DNA sequences without re-engineering the light inducible proteins. This approach took advantage of CRISPR/Cas9 technology, which uses a gene-specific guide RNA (gRNA) to facilitate Cas9 targeting and binding to a desired sequence, and the light-inducible heterodimerizers CRY2 and CIB1 from Arabidopsis thaliana to engineer a light-activated CRISPR/Cas9 effector (LACE) system. We fused the full-length (FL) CRY2 to the transcriptional activator VP64 (CRY2FL-VP64) and the N-terminal fragment of CIB1 to the N-, C-, or N- and C- terminus of a catalytically inactive Cas9. When CRY2-VP64 and one of the CIBN/dCas9 fusion proteins are expressed with a gRNA, the CIBN/dCas9 fusion protein localizes to the gRNA target. In the presence of blue light, CRY2FL binds to CIBN, which translocates CRY2FL-VP64 to the gene target and activates transcription. Unlike other optogenetic systems, the LACE system can be targeted to new endogenous loci by solely manipulating the specificity of the gRNA without having to re-engineer the light-inducible proteins. We achieved light-dependent activation of the IL1RN, HBG1/2, or ASCL1 genes by delivery of the LACE system and four gene-specific gRNAs per promoter region. For some gene targets, we achieved equivalent activation levels to cells that were transfected with the same gRNAs and the synthetic transcription factor dCas9-VP64. Gene activation was also shown to be reversible and repeatable through modulation of the duration of blue light exposure, and spatial patterning of gene expression was achieved using an eGFP reporter and a photomask.

Finally, we engineered a light-activated genetic "on" switch (LAGOS) that provides permanent gene expression in response to an initial dose of blue light illumination. LAGOS is a lentiviral vector that expresses a transgene only upon Cre recombinase-mediated DNA recombination. We showed that this vector, when used in conjunction with a light-inducible Cre recombinase system,1 could be used to express MyoD or the synthetic transcription factor VP64-MyoD2 in response to light in multiple mammalian cell lines, including primary mouse embryonic fibroblasts. We achieved light-mediated upregulation of downstream myogenic markers myogenin, desmin, troponin T, and myosin heavy chains I and II as well as fusion of C3H10T½ cells into myotubes that resembled a skeletal muscle cell phenotype. We also demonstrated LAGOS functionality in vivo by engineering the vector to express human VEGF165 and human ANG1 in response to light. HEK 293T cells stably expressing the LAGOS vector and transiently expressing the light-inducible Cre recombinase proteins were implanted into mouse dorsal window chambers. Mice that were illuminated with blue light had increased microvessel density compared to mice that were not illuminated. Analysis of human VEGF and human ANG1 levels by enzyme-linked immunosorbent assay (ELISA) revealed statistically higher levels of VEGF and ANG1 in illuminated mice compared to non-illuminated mice.

In summary, the objective of this work was to engineer robust light-inducible gene regulation systems that can control genes and cellular fate in a spatial and temporal manner. These studies combine the rapid advances in gene targeting and activation technology with natural light-inducible plant protein interactions. Collectively, this thesis presents several optogenetic systems that are expected to facilitate the development of multicellular cell and tissue constructs for use in tissue engineering, synthetic biology, gene therapy, and basic science both in vitro and in vivo.

The human epidermal growth factor receptor 2, or HER2, is overexpressed in 20-30% breast cancer patients and is associated with aggressive disease. Therapies targeting HER2, including monoclonal antibodies (trastuzumab and pertuzumab), a small molecule kinase inhibitor (lapatinib) and an antibody-drug conjugate (trastuzumab emtansine), have significantly prolonged the overall survival of HER2-positive breast cancer patients. However, almost all patients develop resistance either from the beginning of therapy or with prolonged treatment in two years.

Previous studies to unveil the resistance mechanisms were mainly focused on acquired resistance, culturing cells with HER2 inhibitors and making comparisons to their parental cells. In order to study the mechanism mediating intrinsic resistance, we conducted a loss-of-function genetic screen using a HER2-amplified cell line that is intrinsically resistant to HER2 inhibitors with the purpose to identify synthetic lethal targets. TALDO1, a gene encoding a metabolic enzyme in the non-oxidative pentose phosphate pathway was identified from the screen. Metabolic profiling with isotope-labeled glucose was used to understand the mechanism. The profiling results indicated that TALDO1 was necessary for cellular NADPH generation to combat increased cellular ROS and support synthesis of lipids as a result of HER2 inhibition.

Importantly, the higher expression of TALDO1 is associated with poor response to HER2-targeted therapy in a small cohort of HER2-positive breast cancer patients, suggesting it could potentially serve as a biomarker to predict patient response.

Together our study explained a novel mechanism mediating intrinsic resistance to HER2 inhibition with significant clinical value. Combined inhibition of HER2 signaling and the pentose phosphate pathway may result in a better clinical outcome.

In the last decade, the paradigm for cancer therapy has incrementally transitioned away from non-specific cytotoxic therapies (radiation, chemotherapy) and targeted therapies (small molecules, biologics) and towards immune cell-based therapies. Immune cell-based therapies such as adoptive T cell therapy (ACT) harness the intrinsic ‘sense and respond’ functions of immune cells to selectively target and eliminate cancer cells. Nevertheless, more than half of cancer patients either do not respond or relapse to existing ACTs. Several studies have defined specific transcriptional and epigenetic signatures of the infused T cell product associated with clinical response, indicating that T cell state and fitness is linked to ACT efficacy. Thus, epigenetically reprogramming T cells with enhanced potency and durability has the potential to improve ACT. However, this potential has yet to be fully realized due to technical challenges of adapting CRISPR-based epigenome editing technologies for applications in primary human T cells. To overcome these challenges, we developed and rigorously characterized compact and robust CRISPR repressors and activators for endogenous gene regulation. Next, we leveraged these technologies to systematically interrogate the effects of >100 transcriptional and epigenetic regulators on human CD8+ T cell state and function through complementary CRISPR interference (CRISPRi) and activation (CRISPRa) screens. These CRISPRi/a screens converged on basic leucine zipper ATF-like transcription factor (BATF3). Subsequent assays revealed that BATF3 overexpression promotes specific features of memory T cells (such as increased expression of IL7R and glycolysis), counters T cell exhaustion, and enhances CAR T cell potency in both in vitro and in vivo tumor models. In addition, BATF3 programs a transcriptional profile strongly associated with positive clinical response to CD19 CAR T cell therapy. Given that BATF3 is a compact transcription factor (TF) without any transactivation or epigenetic domains, we speculated that BATF3 achieves its widespread effects by interacting with other TFs. To identify these factors, we conducted parallel CRISPR knockout screens targeting all TFs with or without BATF3 overexpression. Using IL7R expression as a proxy for BATF3 activity, we identified both BATF3-independent and dependent transcriptional regulators of IL7R expression. For example, JUNB and IRF4 were uniquely enriched in the low IL7R population in the screen with BATF3 overexpression, suggesting BATF3 heterodimerizes with JUNB and interacts with IRF4 to regulate gene expression. Finally, these CRISPR knockout screens illuminated other candidate therapeutic targets for future exploration and characterization. Overall, we have developed a widely applicable synthetic biology toolkit of orthogonal epigenome editors, which we used to systematically identify regulators of human CD8+ T cell state and function. This catalogue of regulators could serve as the basis for engineering next generation T cell therapies for cancer.

Basement membranes are a specialized type of extracellular matrix found covering most tissues in animals. These structures are made up of many proteins, most notably laminin and type IV collagen, which form separate polymeric networks that are the core of the BM. BMs are involved in many cell and tissue scale processes during development and homeostasis, and misregulation of BM components lies at the heart of many pathologies. Despite their importance, many of the fundamental aspects of BM biology are not well understood. For example, the mechanisms that regulate differences in BM composition, dynamics, and ultrastructure remain largely unknown. One reason for this is the lack of a model to study these processes in vivo. This has also led to BM dependent processes, such as tissue adhesion through BMs, to be largely overlooked. In Chapter 1, I summarize some of my basic knowledge of BMs, highlight important areas that require further study, and review the process of tissue adhesion through BMs. In Chapter 2, I discuss the creation of an in vivo toolkit of endogenously fluorescently labeled BM components, show how these tools can be used to address questions surrounding BM composition and dynamics, and use these tools to identify papilin as a regulator of type IV collagen network architecture in growing tissues. In Chapter three, I explore the process of tissue adhesion through BMs in greater detail, and identify an enrichment of type IV collagen mediated by tissue specific modifications of the BM that is required to maintain stable BM adhesions between tissues. In Chapter 4, I discuss these findings in more detail, their implications, and future directions based off of this work.

While much of the current focus on advancing CRISPR-Cas9 editing revolves around the engineering of Cas9, the interrogation and evolution of sgRNA scaffold, in addition to novel Cas9 binding RNAs, represent another echelon of development and therapeutic potential. Currently, the majority of research utilizes a singular guide RNA scaffold sequence (the sgRNA) for a given Cas protein (e.g., the Streptococcus pyogenes Cas9 and associated guide RNA). This sequence inflexibility makes many sites within the genome intractable to CRISPR/Cas editing, often due to undesirable intramolecular interactions that result in undesirable secondary structures. Additionally, given the electrostatic potential of Cas9, it may be possible to generate additional Cas9 binding RNA molecules.Here, we use utilize SELEX to both identify novel Cas9 binding RNAs and interrogate the sequence depth of the sgRNA scaffold. First, a SELEX scheme utilizing a nitrocellulose filter binding assay was utilized to identify modified RNA aptamers that bind to Cas9 with specificity and an affinity rivaling that of the sgRNA. The aptamer was shown to tolerate truncations and sequence additions, demonstrating an ability to localize oligonucleotide-based therapeutics to the Cas9 protein. We believe that this aptamer can be expanded upon to incorporate novel functions instead of altering the sgRNA . Second, we use a novel combinatorial approach that utilizes a functional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify numerous, diverse sgRNA variants that bind S. pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in the sgRNA sequence and are utilized in a combinatorial approach to identify scaffolds that enhance editing efficiencies when paired DNA-binding antisense domains. Using molecular evolution, guide RNA scaffolds can be generated for specific targets and optimized to ensure that secondary structure is maintained. This selection approach should be valuable for generating gRNAs with a range of new activities.