Parallel Genetics of Gene Regulatory Sequences in Caenorhabditis elegans

Wie regulatorische Sequenzen die Genexpression steuern, ist von grundlegender Bedeutung für die Erklärung von Phänotypen in Gesundheit und Krankheit. Die Funktion regulatorischer Sequenzen muss letztlich in ihrer genomischen Umgebung und in entwicklungs- oder gewebespezifischen Zusammenhängen verstanden werden. Da dies eine technische Herausforderung ist, wurden bisher nur wenige regulatorische Elemente in vivo charakterisiert. Hier verwenden wir Induktion von Cas9 und multiplexed-sgRNAs, um hunderte von Mutationen in Enhancern/Promotoren und 3′ UTRs von 16 Genen in C. elegans zu erzeugen. Wir quantifizieren die Auswirkungen von Mutationen auf Genexpression und Physiologie durch gezielte RNA- und DNA-Sequenzierung. Bei der Anwendung unseres Ansatzes auf den 3′ UTR von lin-41, bei der wir hunderte von Mutanten erzeugen, stellen wir fest, dass die beiden benachbarten Bindungsstellen für die miRNA let-7 die lin-41-Expression größtenteils unabhängig voneinander regulieren können, mit Hinweisen auf eine mögliche kompensatorische Interaktion. Schließlich verbinden wir regulatorische Genotypen mit phänotypischen Merkmalen für mehrere Gene. Unser Ansatz ermöglicht die parallele Analyse von genregulatorischen Sequenzen direkt in Tieren.How regulatory sequences control gene expression is fundamental for explaining phenotypes in health and disease. The function of regulatory sequences must ultimately be understood within their genomic environment and development- or tissue-specific contexts. Because this is technically challenging, few regulatory elements have been characterized in vivo. Here, we use inducible Cas9 and multiplexed guide RNAs to create hundreds of mutations in enhancers/promoters and 3′ UTRs of 16 genes in C. elegans. We quantify the impact of mutations on expression and physiology by targeted RNA sequencing and DNA sampling. When applying our approach to the lin-41 3′ UTR, generating hundreds of mutants, we find that the two adjacent binding sites for the miRNA let-7 can regulate lin-41 expression largely independently of each other, with indications of a compensatory interaction. Finally, we map regulatory genotypes to phenotypic traits for several genes. Our approach enables parallel analysis of gene regulatory sequences directly in animals

Evaluation and rational design of guide RNAs for efficient CRISPR/Cas9-mediated mutagenesis in Ciona

The CRISPR/Cas9 system has emerged as an important tool for various genome engineering applications. A current obstacle to high throughput applications of CRISPR/Cas9 is the imprecise prediction of highly active single guide RNAs (sgRNAs). We previously implemented the CRISPR/Cas9 system to induce tissue-specific mutations in the tunicate Ciona. In the present study, we designed and tested 83 single guide RNA (sgRNA) vectors targeting 23 genes expressed in the cardiopharyngeal progenitors and surrounding tissues of Ciona embryo. Using high-throughput sequencing of mutagenized alleles, we identified guide sequences that correlate with sgRNA mutagenesis activity and used this information for the rational design of all possible sgRNAs targeting the Ciona transcriptome. We also describe a one-step cloning-free protocol for the assembly of sgRNA expression cassettes. These cassettes can be directly electroporated as unpurified PCR products into Ciona embryos for sgRNA expression in vivo, resulting in high frequency of CRISPR/Cas9-mediated mutagenesis in somatic cells of electroporated embryos. We found a strong correlation between the frequency of an Ebf loss-of-function phenotype and the mutagenesis efficacies of individual Ebf-targeting sgRNAs tested using this method. We anticipate that our approach can be scaled up to systematically design and deliver highly efficient sgRNAs for the tissue-specific investigation of gene functions in Ciona

Advanced CRISPR-Cas9 techniques for modulation of non-coding disease-associated genetic variants

The search for genetic explanations of individual susceptibility to complex polygenic diseases has greatly intensified in recent years, with GWAS studies having successfully linked several hundred thousand genomic loci to complex diseases. However, the disease mechanisms underlying these associations remain largely unknown due to several limitations of GWAS. In particular, since 90 % of identified disease-associated SNPs are located outside of protein-coding regions, their biological effects are largely unclear, and although they likely affect gene regulatory functions via altered DNA motifs for specific transcription factors, the target genes need to be identified. Furthermore, each locus contains up to several hundred SNPs in linkage disequilibrium, and thus the causal SNPs are unknown. Recent progress in addressing the problem has been made through the development of a more systematic approach involving several bioinformatic and experimental advances, that specifically tackles the mechanistic limitations of GWAS. The approach applies a series of methods to systematically narrow down the number of candidate causal SNPs, and ultimately identify the causal SNP and affected cell types, enhancers and gene regulatory mechanisms.. To experimentally validate causal SNPs in cellulo and establish downstream target genes and phenotypes, genome editing of the causal SNP must be performed. While regular CRISPR/Cas9 theoretically can be used for this purpose, it is highly inefficient and introduces several issues such as double-stranded breaks and potential off-target effects. In contrast, the newly developed Prime Editing (PE) technology may prove to be ideal for this type of precise genome editing. Furthermore, a second method, CRISPR/Cas9-mediated enhancer modulation (CA/I), may be used to strengthen the findings through direct epigenetic activation or repression of the enhancer in which the SNP resides. As a proof-of-concept study of these recent advances, this thesis builds upon previous unpublished work from our group, which identified a likely causal SNP (rs1799993) in an enhancer associated with visceral obesity, a particularly harmful type of fat accumulation. Epigenetic data suggested that the SNP affects an enhancer element that is active in adipose-derived mesenchymal stem cells (AD-MSCs). Thus, the current study has focused on establishing the genome editing tool PE for in situ editing of the SNP, as well as the epigenetic modulation system CA/I for modulation of the surrounding predicted enhancer element, for use in AD-MSCs. Spacers for sgRNAs and pegRNAs (the latter in PE) targeting the enhancer region and the SNP, respectively, were designed by in silico analysis, and evaluated in vitro for on-target efficiency. Extensions for pegRNAs were designed for editing the SNP from risk to protective allele and vice versa, and a plasmid library of the sgRNAs and pegRNAs was then prepared and sequence-validated. Furthermore, an appropriate mesenchymal stem cell (MSC) model was obtained and genotyped for the SNP in question. Because the MSCs are notoriously hard to transfect, comprehensive testing of transfection methods in these cells was performed, including a variety of chemical and physical methods of gene delivery. While the required plasmids for both PE and CA/I were successfully made, no transfection method proved successful in MSCs using the large-size PE plasmids. Consequently, the SNP rs1799993 was not edited. However, nucleofection was identified as the method that gave the best results in this cell type using smaller plasmids, thus suggesting that optimizations should be directed toward reducing the PE-plasmid size for successful use of this method is MSCs. For similar reasons, a pilot lentiviral transduction of plasmids for CRISPR activation/repression did not result in stably transduced MSCs. In summary, the work of this thesis has laid the groundwork for utilizing the PE and CA/I methods as tools to help translate GWAS association signals into causal gene regulatory mechanisms. Once optimized, the techniques should be able to determine whether rs1799993 is a causal SNP in the visceral obesity associated locus of interest, as well as identify the target genes of the enhancer where the SNP resides and alters transcription factor binding.Masteroppgave i molekylærbiologiMOL399MAMN-MO

CRISPR/Cas9 genome editing of Recessive Dystrophic Epidermolysis Bullosa (RDEB) mutation hotspot

Recessive dystrophic epidermolysis bullosa (RDEB) is a severe life-threatening skin adhesion disorder caused by loss-of-function mutations in the COL7A1-encoding type VII collagen (C7), a structural protein playing a crucial role in anchoring fibril (AF) formation at the dermal-epidermal junction (DEJ). Combinatorial cell and gene therapies based on the addition of a full-length copy of COL7A1 cDNA in RDEB keratinocytes, fibroblasts and skin equivalents have shown potential in preclinical and clinical settings although only modest and transient improvements have been reported. In parallel, induced pluripotent stem cells (iPSCs) are being investigated in preclinical studies for RDEB. iPSCs represent a valuable source of autologous patient material and can be differentiated into keratinocytes and fibroblasts for cellular therapy applications. Implementation of CRISPR/Cas9 and base editing-mediated gene correction in patient-derived iPSCs has allowed for the generation of autologous cellular models capable of overcoming barriers of conventional gene therapy. In this regards, the work described in this thesis aims to evaluate the feasibility of genome-editing approaches using CRISPR/Cas9 and Cytosine Base editing (BE) platforms to correct a mutation hotspot (c.425A>G, p.Lysl42Arg) within exon 3 of COL7A1 gene in patient-derived iPSCs. Gene repair by homology-directed recombination (HDR) following CRISPR/Cas9-induced double-strand breaks (DSBs) through viral and non-viral donor template deliveries resulted in a significant correction of the COL7A1 locus on genomic level. To avoid concerns surrounding the generation of DSBs, seamless BE-based G:C to A:T conversion resulted in a high restoration of the wild type COL7A1 sequence. Ultimately, capacity of gene- and base-corrected RDEB iPSCs to be differentiated in into keratinocytes (iKer) was evaluated in vitro and functional recovery of de novo C7 was assayed on protein level. Overall, this study explored the potential of CRISPR/Cas9 and BE site-specific correction of COL7A1 in RDEB-derived pluripotent stem cells. Furthermore, it demonstrated that gene-corrected iPSCs can be used as a source of epidermal progenitors thereby confirming their potential for future cell therapies for skin disorders

Emerging Gene-Editing Modalities for Osteoarthritis

Osteoarthritis (OA) is a pathological degenerative condition of the joints that is widely prevalent worldwide, resulting in significant pain, disability, and impaired quality of life. The diverse etiology and pathogenesis of OA can explain the paucity of viable preventive and disease-modifying strategies to counter it. Advances in genome-editing techniques may improve disease-modifying solutions by addressing inherited predisposing risk factors and the activity of inflammatory modulators. Recent progress on technologies such as CRISPR/Cas9 and cell-based genome-editing therapies targeting the genetic and epigenetic alternations in OA offer promising avenues for early diagnosis and the development of personalized therapies. The purpose of this literature review was to concisely summarize the genome-editing options against chronic degenerative joint conditions such as OA with a focus on the more recently emerging modalities, especially CRISPR/Cas9. Future advancements in novel genome-editing therapies may improve the efficacy of such targeted treatments

In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering.

RNA regulatory elements (RREs) are an important yet relatively under-explored facet of gene regulation. Deciphering the prevalence and functional impact of this post-transcriptional control layer requires technologies for disrupting RREs without perturbing cellular homeostasis. Here we describe genome-engineering based evaluation of RNA regulatory element activity (GenERA), a clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 platform for in situ high-content functional analysis of RREs. We use GenERA to survey the entire regulatory landscape of a 3'UTR, and apply it in a multiplex fashion to analyse combinatorial interactions between sets of miRNA response elements (MREs), providing strong evidence for cooperative activity. We also employ this technology to probe the functionality of an entire MRE network under cellular homeostasis, and show that high-resolution analysis of the GenERA dataset can be used to extract functional features of MREs. This study provides a genome editing-based multiplex strategy for direct functional interrogation of RNA cis-regulatory elements in a native cellular environment