With advancements in CRISPR-cas9 broadening the potential paths for clinical usage of genetic editing, conversations about genetic editing have grown to outside simply scientific communities and into mainstream conversations. This study focuses specifically on Christian discourse of genetic editing and locates four major tensions for many Christians when they think about genetic editing: beginning of life, Creator-human relationship, imago Dei, and stewardship. With these major concerns in mind, I identify epigenetics, somatic cell genetic editing, and in vivo genetic editing research as important research paths to pursue as they can potentially produce techniques that more Christian individuals would feel comfortable using. I pursue one of these paths and conclude with an experimental proposal for an analysis of in vivo and in vitro CIRSPR-Cas9 efficiency in regards to on- and off-target rates.

Les CRISPRs constituent une famille particulière d'éléments génétiques, retrouvés dans de nombreux génomes de procaryotes (la moitié des bactéries et presque toutes les archées). Des études récentes suggèrent que cette structure représente un système de défense contre les ADNs étrangers fonctionnant grâce à un mécanisme d'interférence ARN. Les CRISPRs consistent en la succession de régions très bien conservées (DR) dont la taille varie de 23 à 47 pb, séparées par des séquences uniques d'une taille similaire et ayant en général une origine virale. Le polymorphisme observé entre différentes souches de la même espèce fait du CRISPR un marqueur génétique intéressant pour des analyses comparatives de souches bactériennes très proches et pour des études phylogénétiques. Cette thèse décrira trois outils bioinformatiques accessibles à l'adresse (http://crispr.u-psud.fr) et leurs applications dans l'investigation et le typage des CRISPRs. Le premier outil est CRISPRFinder qui est un programme d'identification des CRISPRs à partir des séquences génomiques. Le deuxième est la base de données CRISPRdb et ses utilitaires qui fournissent un accès aux CRISPRs de tous les génomes procaryotes séquencés. le troisième outil est CRISPRcompar qui sert à identifier et comparer les CRISPRs dans des génomes proches pour faciliter la procédure de typage.

Bacterial pathogens rely on precise regulation of gene expression to coordinate host infection processes and resist invasion by mobile genetic elements. An interconnected network of protein and RNA regulators dynamically controls the expression of virulence factors using a variety of mechanisms. In this thesis, the role of selected regulators, belonging to the class of small RNAs (sRNAs), is investigated. Streptococcus pyogenes is a pathogen responsible for a wide range of human diseases. Genome-wide screenings have indicated that S. pyogenes encodes numerous sRNAs, yet only a limited number have been characterized. A major goal of this study was to identify and characterize novel sRNAs and antisense RNAs (asRNAs) using RNA sequencing analysis. We validated 30 novel sRNAs and asRNAs, and identified 9 sRNAs directly cleaved by the ribonucleases RNase III and/or RNase Y. Previous work from the laboratory has highlighted the role of sRNAs from the type II Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated proteins (CRISPR-Cas) systems in S. pyogenes. CRISPR-Cas systems provide adaptive immunity to prokaryotes against infection by mobile genetic elements. Two sRNAs, forming a complementary duplex (dual-RNA), are effectors of this system: the mature CRISPR RNAs (crRNAs) and the trans-activating crRNA (tracrRNA). The dual-RNA guides the Cas9 endonuclease to cleave both strands of the invading DNA in a sequence-specific manner. This RNA-programmable CRISPR-Cas9 system is now utilized for genome editing and engineering in a wide range of cells and organisms. To expand the potentialities of this tool, we both, searched for Cas9 orthologs and predicted numerous tracrRNA orthologs. We defined tracrRNA as a new family of sRNAs sharing the ability to base-pair to cognate crRNAs, without conservation of structure, sequence or location. We show that Cas9 and the dual tracrRNA:crRNAs are only interchangeable between closely related type II CRISPR-Cas systems. In summary, this thesis presents new insights into RNA-mediated regulatory mechanisms in S. pyogenes. We identified and described the expression of novel sRNAs, highlighting potential antisense RNAs. Focusing on the dual-RNA programmable type II CRISPR-Cas system, we provided evidence for co-evolution of the Cas9 enzyme with tracrRNA:crRNA, a basis for Cas9 multiplexing in genome editing.

Proteoglycans (PGs) are composed of highly sulfated glycosaminoglycans chains (GAGs) attached to specific core proteins. They are present in extracellular matrices, on the cell surface and in storage granules of hematopoietic cells. Heparan sulfate (HS) and chondroitin/dermatan sulfate (CS/DS) GAGs play indispensable roles in a wide range of biological processes, where they can serve as protein carriers, be involved in growth factor or morphogen gradient formation and act as co-receptors in signaling processes. Protein binding abilities of GAGs are believed to be predominantly dependent on the arrangement of the sugar modifications, sulfation and epimerization, into specific oligosaccharide sequences. Although the process of HS and CS/DS assembly and modification is not fully understood, a set of GAG biosynthetic enzymes have been fairly well studied and several mutations in genes encoding for this Golgi machinery have been linked to human genetic disorders. This thesis focuses on the zebrafish N-deacetylase/N-sulfotransferase gene family, encoding key enzymes in HS chain modification, as well as glycosyltransferases responsible for chondroitin/dermatan sulfate elongation present in zebrafish. Our data illustrates the strict spatio-temporal expression of both the NDST enzymes (Paper I) and CS/DS glycosyltransferases (Paper II) in the developing zebrafish embryo. In Paper III we took advantage of the four preexisting zebrafish mutants with defective GAG biosynthesis. We could demonstrate a relation between HS content and the severity of the pectoral fin defects, and additionally correlate impaired HS biosynthesis with altered chondrocyte intercalation. Interestingly, altered CS biosynthesis resulted in loss of the chondrocyte extracellular matrix. One of the main findings was the demonstration of the ratio between the HS biosynthesis enzyme Extl3 and the Csgalnact1/Csgalnact2 proteins, as a main factor influencing the HS/CS ratio. In Paper IV we used the newly developed CRISPR/Cas9 technique to create a collection of zebrafish mutants with defective GAG biosynthetic machineries. Lack of phenotypes linked to null-mutations of most of the investigated genes is striking in this study.

The human genome encodes information that instructs human development, physiology, medicine, and evolution. Massive amount of genomic data has generated an ever-growing pool of hypothesis. Genome editing, broadly defined as targeted changes to the genome, posits to deliver the promise of genomic revolution to transform basic science and personalized medicine. This thesis aims to contribute to this scientific endeavor with a particular focus on the development of effective human genome engineering tools.

The advancement of high-throughput, large-scale biochemical, biophysical, and genetic technologies has enabled the generation of massive amounts of biological data and allowed us to synthesize various types of biomaterial for engineering purposes. This enabled improved observational methodologies for us to navigate and locate, with unprecedented resolution, the potential factors and connections that may contribute to biological and biomedical processes. Nonetheless, it leaves us with the increasing demand to validate these observations to elucidate the actual causal mechanisms in biology and medicine. Due to the lack of powerful and precise tools to manipulate biological systems in mammalian cells, these efforts have not been able to progress at an adequate pace.

The dynamic nature of gene expression enables cellular programming, homeostasis and environmental adaptation in living systems. Dissection of causal gene functions in cellular and organismal processes therefore necessitates approaches that enable spatially and temporally precise modulation of gene expression. Recently, a variety of microbial and plant-derived light-sensitive proteins have been engineered as optogenetic actuators, enabling high-precision spatiotemporal control of many cellular functions. However, versatile and robust technologies that enable optical modulation of transcription in the mammalian endogenous genome remain elusive. Here we describe the development of light-inducible transcriptional effectors (LITEs), an optogenetic two-hybrid system integrating the customizable TALE DNA-binding domain with the light-sensitive cryptochrome 2 protein and its interacting partner CIB1 from Arabidopsis thaliana. LITEs do not require additional exogenous chemical cofactors, are easily customized to target many endogenous genomic loci, and can be activated within minutes with reversibility. LITEs can be packaged into viral vectors and genetically targeted to probe specific cell populations. We have applied this system in primary mouse neurons, as well as in the brain of freely behaving mice in vivo to mediate reversible modulation of mammalian endogenous gene expression as well as targeted epigenetic chromatin modifications. We explore the modularity of the LITE approach through the development of CRISPR/Cas9 transcriptional effectors in either constitutively active or light-inducible contexts. The LITE system establishes a novel mode of optogenetic control of endogenous cellular processes and enables direct testing of the causal roles of genetic and epigenetic regulation in normal biological processes and disease states. / Engineering and Applied Sciences

Recent advances in genome engineering technologies based on the CRISPR-associated RNA-guided endonuclease Cas9 are enabling the systematic interrogation of genome function. Analogous to the search function in modern word processors, Cas9 can be guided to specific locations within complex genomes by a short RNA search string. Using this system, DNA sequences within the endogenous genome and their functional outputs are now easily edited or modulated in virtually any organism of choice. Cas9-mediated genetic perturbation is simple and scalable, empowering researchers to elucidate the functional organization of the genome at the systems level and establish causal linkages between genetic variations and biological phenotypes. To facilitate successful and specific Cas9 targeting, we first optimize the guide RNAs (sgRNA) to significantly enhance gene editing efficiency and consistency. We also systematically characterize Cas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target mutagenesis. We find that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. We also show that Cas9-mediated cleavage is unaffected by DNA methylation and that the dosage of Cas9 and sgRNA can be titrated to minimize off-target modification. Additionally, we provide a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses. We next demonstrate that Cas9 nickase mutants can be used with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs can reduce off-target activity by over 1,500-fold in human cells. In collaboration with researchers at the University of Tokyo, we further identified a PAM-interacting domain of the Cas9 nuclease that dictates Cas9 target recognition specificity. Finally, we present protocols that provide experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks. Taken together, this work enables a variety of genome engineering applications from basic biology to biotechnology and medicine.

Developing targeted genome regulation approaches holds much promise for accelerating trait discovery and development in agricultural biotechnology. Clustered Regularly Interspaced Palindromic Repeats (CRISPRs)/CRISPR associated (Cas) system provides bacteria and archaea with an adaptive molecular immunity mechanism against invading nucleic acids through phages and conjugative plasmids. The type II CRISPR/Cas system has been adapted for genome editing purposes across a variety of cell types and organisms. Recently, the catalytically inactive Cas9 (dCas9) protein combined with guide RNAs (gRNAs) were used as a DNA-targeting platform to modulate the expression patterns in bacterial, yeast and human cells. Here, we employed this DNA-targeting system for targeted transcriptional regulation in planta by developing chimeric dCas9-based activators and repressors. For example, we fused to the C-terminus of dCas9 with the activation domains of EDLL and TAL effectors, respectively, to generate transcriptional activators, and the SRDX repression domain to generate transcriptional repressor. Our data demonstrate that the dCas9:EDLL and dCas9:TAD activators, guided by gRNAs complementary to promoter elements, induce strong transcriptional activation on episomal targets in plant cells. Moreover, our data suggest that the dCas9:SRDX repressor and the dCas9:EDLL and dCas9:TAD activators are capable of markedly repressing or activating, respectively, the transcription of an endogenous genomic target. Our data indicate that the CRISPR/dCas9:TFs DNA targeting system can be used in plants as a functional genomic tool and for biotechnological applications.

Asthma is a major public health epidemic that presents a heavy burden on those who suffer from the disease. Little is currently understood about the genetic signature that distinguishes one type of asthma from another. Recently, the single nucleotide polymorphism (SNP) rs968567 was found to have a high degree of association in asthmatic patients (Sharma et al., 2014). This particular SNP is in the promoter region of the FADS2 gene that synthesizes the enzyme delta-6-desaturase (D6D). D6D mediates the formation of pro-inflammatory factors that lead to exacerbation of asthmatic symptoms. We engineered a novel, customized CRISPR-Cas9 construct to induce the SNP rs968567 in the HUES9 human embryonic stem cell (hESC) line. Our results show success in generating the custom CRISPR-Cas9 construct for use in stem cells, while efficiency in expressing the desired mutation in our cell line is currently being optimized. Disease modeling in the genomic era of medicine provides an opportunity for the development of personalized medical treatment. Future projects aim to differentiate stem cell lines edited with our CRISPR-Cas9 construct to lung progenitor cells to study the cellular phenotype of this mutation in context of asthma pathogenesis.