Site specific modification of the genome represents a powerful tool to investigate the functional consequences of DNA sequence mutation and variation. Techniques, which enable specific nucleotides to be precisely modified, facilitate the generation of disease models harbouring pathogenic human mutations. These models can be used to explore the biological underlying the disease process and may also serve as a resource for therapeutic approaches. Genome Wide Association Studies (GWAS) and resequencing experiments are revealing sequence variants that associate with disease susceptibility. Tools enabling the experimental introduction of putative disease SNPs into the genome represent a vital validation tool to commence the functional dissection of this vast data set.
Traditional methods of gene manipulation have relied on homologous recombination between the chromosome and purpose build DNA constructs, frequently in stem cells. These cells can then be used to generate disease models or can be monitored for functional consequences, e.g. gene expression changes in vitro.
New technology has become available which allows the manipulation or editing of the genome to be achieved at high efficiency, using nucleases known as TALENs (Davies et al., 2013) or CRISPR (Wang et al. 2013). Site specific nucleases can now easily be constructed against specific genomic target sites and can be used either within stem cells or oocytes to introduce specific double strand breaks within the genome. These double strand breaks are repaired by the cellular machinery, which can lead to the introduction of mutations or (if a template is presented to the cell) the introduction of specific nucleotide changes (Yang et al., 2013).
This project aims at examining the application of site specific nuclease technology to achieve precise genome editing in model systems, and will make use of CRISPR based technologies. Enzymes will be designed against specific sequences and used to introduce disease relevant SNPs into the genome. The tools developed in this project will enable an understanding of how mutation can influence specific disease susceptibility.
The research project will thus focus on the technological aspects of achieving site specific genome modification (“genome editing”), appropriate delivery and screening methods, as well as exploring potential off-target effects. However, we will be able to take advantage of the disease relevant SNPs that have been identified within the centre as contributing to increased susceptibility to a variety of diseases, such as cancer, cardiovascular disease and diabetes and thus to explore this new technology for the generation of novel disease models. Close collaboration between our technology focussed core group and one of the disease groups within the centre is anticipated.
Project reference number: 758
|Dr Ben Davies||Wellcome Trust Centre for Human Genetics||Oxford University, Henry Wellcome Building of Genomic Medicine||GBRfirstname.lastname@example.org|
|Prof Mark McCarthy||OCDEM||Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism||GBRemail@example.com|
Human pluripotent stem cells (hPSCs) offer unprecedented opportunities to study cellular differentiation and model human diseases. The ability to precisely modify any genomic sequence holds the key to realizing the full potential of hPSCs. Thanks to the rapid development of novel genome editing technologies driven by the enormous interest in the hPSC field, genome editing in hPSCs has evolved from being a daunting task a few years ago to a routine procedure in most laboratories. Here, we provide an overview of the mainstream genome editing tools, including zinc finger nucleases, transcription activator-like effector nucleases, clustered regularly interspaced short palindromic repeat/CAS9 RNA-guided nucleases, and helper-dependent adenoviral vectors. We discuss the features and limitations of these technologies, as well as how these factors influence the utility of these tools in basic research and therapies. Hide abstract
Transcription Activator-Like Effector Nucleases (TALENs) consist of a nuclease domain fused to a DNA binding domain which is engineered to bind to any genomic sequence. These chimeric enzymes can be used to introduce a double strand break at a specific genomic site which then can become the substrate for error-prone non-homologous end joining (NHEJ), generating mutations at the site of cleavage. In this report we investigate the feasibility of achieving targeted mutagenesis by microinjection of TALEN mRNA within the mouse oocyte. We achieved high rates of mutagenesis of the mouse Zic2 gene in all backgrounds examined including outbred CD1 and inbred C3H and C57BL/6J. Founder mutant Zic2 mice (eight independent alleles, with frameshift and deletion mutations) were created in C3H and C57BL/6J backgrounds. These mice transmitted the mutant alleles to the progeny with 100% efficiency, allowing the creation of inbred lines. Mutant mice display a curly tail phenotype consistent with Zic2 loss-of-function. The efficiency of site-specific germline mutation in the mouse confirm TALEN mediated mutagenesis in the oocyte to be a viable alternative to conventional gene targeting in embryonic stem cells where simple loss-of-function alleles are required. This technology enables allelic series of mutations to be generated quickly and efficiently in diverse genetic backgrounds and will be a valuable approach to rapidly create mutations in mice already bearing one or more mutant alleles at other genetic loci without the need for lengthy backcrossing. Hide abstract
The type II bacterial CRISPR/Cas system is a novel genome-engineering technology with the ease of multiplexed gene targeting. Here, we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA and different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one-step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2, and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances. Hide abstract