CRISPR-activation and inhibition - manipulating gene expression within the stem cell

Project Overview

Induced pluripotent stem cells

Upregulation of a target gene by CRISPRa

CRISPR/Cas9 is a programmable site-specific nuclease that can be used to manipulate the sequence of DNA in a precise and efficient manner. Using these “genome editing” approaches, genes can be knocked out of the genome to allow an assessment of their function in both stem cell and animal models. Furthermore, single nucleotide variations which have been found to associate with a particular disease progression or susceptibility can also be introduced into these systems, allowing models of human disease to be interrogated within the laboratory.

As well as being an effective genome-editing tool, the CRISPR/Cas9 system, if rendered catalytically inactive, serves as a very effective programmable site-specific DNA binding protein. Using this system, effector domains which can either up- or down-regulate gene expression can be recruited to gene promoters and regulatory regions, allowing tuneable manipulation of gene expression levels – so called CRISPRa (activation) and CRISPRi (inactivation) technology.

Since it is becoming clear that much of the genomic variation which inlfuence disease, lies within regulatory regions of the genome, the development of robust systems for manipulating gene expression in models systems will enable insights into how perturbations in gene expression networks underlie changes in disease susceptibility and progression.

This research project will focus on efficient methods of implementing CRISPRa and CRISPRi in human induced pluripotent stem (iPS) cells, allowing perturbations in gene expression from endogenous promoters in human cells. These iPS cells can be differentiated into disease-relevant cell types and the consequence of gene expression changes monitored using phenotypic assays. Modulating the expression of key transcription factors involved in differentiation may also allow new protocols for efficient stem-cell differentiation to be developed. Close collaboration between our technology focussed core group and one of the disease groups within the centre is anticipated.

Training Opportunities

Theme

Genetics & Genomics and Physiology, Cellular & Molecular Biology

Admissions

Project reference number: 758

Funding and admissions information

Supervisors

Name Department Institution Country Email
Associate Professor Ben Davies Wellcome Trust Centre for Human Genetics Oxford University, Henry Wellcome Building of Genomic Medicine GBR ben.davies@well.ox.ac.uk
Prof Mark McCarthy OCDEM Oxford University, Oxford Centre for Diabetes, Endocrinology & Metabolism GBR mark.mccarthy@drl.ox.ac.uk

Dominguez AA, Lim WA, Qi LS. 2016. Beyond editing: repurposing CRISPR-Cas9 for precision genome regulation and interrogation. Nat. Rev. Mol. Cell Biol., 17 (1), pp. 5-15. Read abstract | Read more

The bacterial CRISPR-Cas9 system has emerged as a multifunctional platform for sequence-specific regulation of gene expression. This Review describes the development of technologies based on nuclease-deactivated Cas9, termed dCas9, for RNA-guided genomic transcription regulation, both by repression through CRISPR interference (CRISPRi) and by activation through CRISPR activation (CRISPRa). We highlight different uses in diverse organisms, including bacterial and eukaryotic cells, and summarize current applications of harnessing CRISPR-dCas9 for multiplexed, inducible gene regulation, genome-wide screens and cell fate engineering. We also provide a perspective on future developments of the technology and its applications in biomedical research and clinical studies. Hide abstract

Li M, Suzuki K, Kim NY, Liu GH, Izpisua Belmonte JC. 2014. A cut above the rest: targeted genome editing technologies in human pluripotent stem cells. J. Biol. Chem., 289 (8), pp. 4594-9. Read abstract | Read more

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