Gene editing and gene regulation with CRISPR

Abstract

Sixty years ago, Francis Crick put forward his hypothesis on the mechanism for the flow of information from DNA, via RNA, to protein, a process which became known as the central dogma of molecular biology. From an experimental point of view, this means that if we have techniques that can modify the sequence of DNA efficiently and precisely, we can explore experimentally the physiological consequences of this for the cell and, ultimately, the organism. To help explain the range of techniques available to modify or edit DNA sequences in living cells, the technical challenges and emerging solutions, and the application of these techniques to gain a better understanding of physiological processes and, potentially, treat disease, the Physiological Society invited a group of local and international experts to take part in the symposium ‘Gene Editing and Gene Regulation with CRISPR’, which was held at Hodgkin Huxley House in London on 15 November 2016.

To get things started, Stephen Hart (University College London) took to the floor to introduce the speakers in the first of four sessions. First up was Patrick Harrison (University College Cork), who gave a brief history of gene editing and the development of the three major editing nucleases, ZFNs TALENs and CRISPR (Harrison & Hart, 2018). Having set the scene, Claudio Mussolino (University Medical Center Freiburg) took the stage to describe the use of TALENs and other nucleases to edit bone marrow-derived cells precisely for the potential treatment of primary immune disorders. These rare genetic disorders are seen as one of the first potential targets for therapeutic gene editing, because the target cells can be extracted from a patient's bone marrow, edited ex vivo and transplanted back into the patient. With the defect corrected by gene editing, these autologous cells should not be rejected by the patient, as the patient is also the donor! To close the opening session, Rakhi Harne (University of Edinburgh) gave the first of the selected abstract talks describing the use of CRISPR gene editing to modify the protein tyrosine kinase receptor, CSF1R, in chicken primordial germ cells.

After coffee, Rameen Shakur (University of Cambridge) gave a talk demonstrating the power of CRISPR gene editing to generate isogenic human inducible pluripotent stem (iPS) cells; when differentiated into cardiac myocytes, these cells enabled him to model sudden cardiac death in hypertrophic cardiomyopathy, and he discussed the implications for personalized medicine. This was followed by a talk on the gene editing of skeletal muscle by Linda Popplewell (Royal Holloway University of London), in the context of the development of therapeutic strategies for muscular dystrophies. This talk also introduced the cross-over between gene editing (precise modification of a small number of nucleotides) and gene addition (the introduction of large DNA sequences). Session two was drawn to a close with two talks selected from submitted abstracts by Iffath Ghouri (Newcastle University), who described the pitfalls encountered as a gene editing novice when targeting and validating knockout of lysine deacetylase enzymes, and Toby Collins (University College London), who was investigating the functional consequences of IFT80 mutations in Jeune syndrome using CRISPR/Cas9.

After a very interactive lunchbreak with extensive discussion about gene editing and a surfeit of poor jokes about the CRISPS served with the delicious CUT sandwiches, we moved on to the third session, with two talks about the development of gene editing as potential therapies for rare diseases. First, Julia Reichelt (University Hospital, Salzburg) showed the potential of gene editing to correct mutations in epithelial cells in the context of rare skin disorders (March, Reichelt & Koller, 2018). This was followed by Ciaran Lee (Rice University, TX, USA) who described gene editing for sickle cell anaemia and the development of techniques to reduce off-target effects of gene editing (Lee, Davis, & Bao, 2018). This session was concluded by two more selected abstracts, one by Nagendra Babu Thillaiappan (University of Cambridge), who showed in exquisite detail how gene editing could be used to label IP₃ receptors with fluorescent markers and revealed that immobile IP₃ receptors at the endoplasmic reticulum–plasmalemmal junction could initiate Ca²⁺ signals, and another from Karin Tuschl (University College London and King's College London), who described how genome editing is performed in zebrafish to elucidate mechanisms of inborn errors of metabolism.

The first talk of the final session, by Kristian Skipper (Aarhus University), tackled a key issue in the field, the delivery of genomic engineering and editing tools, including the development of viral vectors that could deliver gene editing nucleases. This was followed by Rowan Flynn (University of Oxford), who described the editing of iPS cells and their subsequent differentiation to enable the study neurological conditions. The session was closed by Ahmad Aldossary (University College London), with a selected abstract talk on the development of CRISPR/Cas9 to correct the ΔF508 mutation in CFTR, which causes cystic fibrosis.

In summary, the symposium provided an excellent introduction to the field of gene editing, with descriptions of the most recent developments in gene editing tools and delivery methods to edit a diverse range of cells and animals to generate isogenic controls in modelling both physiological and disease processes. Future challenges will include the refinement of the technology to minimize off-target effects, optimize on-target efficiency and improve in vivo delivery strategies. It is also likely that Cas9 variants that can modulate gene expression or edit individual base pairs without the need to cut the DNA backbone will become more widely used. Given the unprecedented power of gene editing to modify the genome, a greater focus on the scientific and public discussion on the ethical and regulatory issues surrounding how these techniques will be used is also important.