CRISPR Chemistry

Lead Research Organisation: University of Oxford
Department Name: Oxford Chemistry

Abstract

A transformative new technology for gene editing, known as CRISPR-Cas, was recently discovered. It allows a protein (Cas9) to be programmed by a CRISPR RNA molecule to modify specific sequences in genomic DNA. This can be used to alter the levels of specific proteins in cells, and can even be used to eliminate them or change their function (via site-specific mutation). This technology has profound implications in many areas of biology and medicine and, in the future, could be used to successfully treat human genetic diseases and cancer. By creating artificial (modified) Cas9 proteins, it is also possible to edit the cell's RNA molecules and to create detailed images of the genome in live cells in real-time. However, there are drawbacks to CRISPR-Cas technology: the number of CRISPR RNA molecules needed to perform some editing tasks is cost-prohibitive, imaging multiple DNA loci in the genome is difficult, and to completely change a cell's characteristics (cell type) is not currently feasible. Moreover, unintended editing events in the genome (off-target effects) are common and these are potentially catastrophic, particularly in the therapeutic arena where they could cause cancer when used in therapeutic applications. We will address these problems by chemically modifying the Cas9-binding CRISPR RNA molecule in various ways. Libraries of RNA molecules will be created by mixing and matching (chemically ligating) the short variable gene-targeting RNA moiety with a larger invariable RNA sequence needed for association with Cas9 protein. By adding fluorescent dyes to these RNA molecules and developing systems that intelligently activate and programme the output colour, live-cell imaging capabilities will be significantly improved. In order to control a cell's behaviour, we will append short pieces of DNA to the CRISPR RNA molecule that will recruit key gene regulating proteins within the cell and enable us to fine-tune gene expression. Finally, the use of light-activated chemically modified Cas9-binding RNA molecules, which can only transiently and subtly damage DNA, will be investigated to reduce undesirable off-target effects. These nucleic acid chemistry-driven objectives are aimed at facilitating research that would otherwise be impractical or impossible using current Cas9 technology and in the long term could enable exciting new applications.

Planned Impact

The results of this project will be of benefit to researchers working in biology, genetics, biomedical science and oncology as it will provide new tools that will improve the efficiency of gene editing/regulation and facilitate fluorescent imaging of specific motifs in genomic DNA in live cells. It will also encourage further research in nucleic acid chemistry and stimulate the growth of this field in the UK. The interdisciplinary training that the postdoctoral researcher employed on this project will receive will provide her/him with a broad set of skills, making them highly sought after. This project is relevant to the following EPSRC research areas: biophysics (quantitative, experimental approaches with biological questions and hypotheses), chemical biology and biological chemistry (development of novel chemical tools and technologies for the understanding of biology and the synthesis of biologically active molecules, biomimetic chemistry, synthetic methods that mimic biochemical processes); medical imaging (for therapeutic, monitoring and diagnostic purposes); synthetic biology (to design and engineer novel biologically-based parts, devices and systems that do not exist in the natural world). Chemical biology and biophysics are two of the three areas of growth in the EPSRC Physical Science portfolio.
Economic
The development of new scalable chemical approaches to modified CRISPR RNA will overcome some of the key constraints of current gene-editing technologies. This will have an impact in the fields of biotechnology and therapeutics. It will provide new IP and licencing opportunities for Oxford University which will be pursued through interactions with Oxford Innovation and via presentations at conferences with high level industrial participation, as well as by direct approaches to Pharma and Biotech companies with interests in the diagnostic and therapeutic fields. The commercial potential is very significant and practical applications are not restricted to humans; CRISPR has applications in animal health and in plant science.
Societal
This generic technology has potential applications in many areas, one of which is the cancer field. New and improved therapeutic CRISPR-based approaches in oncology could have a major impact on society in terms of cost, quality of life and survival rates. Therapies based on CRISPR are also relevant to ageing and genetic diseases; many of which have correlations with multiple gene mutations. For example Parkinson's disease, which is caused by neuronal death, is associated with mutations in the PARK2, DJ-1, PINK1, LRRK2 and SNCA genes. Improved CRISPR knockout methods could reveal how such genes interact with each other, aiding in the discovery of druggable targets. Early-onset Alzheimer's disease is associated with the gene encoding amyloid precursor protein (APP), and CRISPR has been shown to correct this mutation in human fibroblast cells. Homology-directed DNA repair initiated by CRISPR has also been shown in model systems to reduce the amyloid plaque formation that is symptomatic of Alzheimer's disease. Haematological genetic disorders such as sickle cell anaemia are also potentially amenable to treatment with CRISPR. The key point is that current CRISPR-based systems have great potential but are not yet suitable for therapeutic applications due to a number of reasons including off-target effects, so the societal impact of advances coming from this project is potentially great.

Publications

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Shivalingam A (2020) Squaramides and Ureas: A Flexible Approach to Polymerase-Compatible Nucleic Acid Assembly. in Angewandte Chemie (International ed. in English)

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Taemaitree L (2020) Shining Light on CRISPR Gene Editing. in ACS central science

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Taemaitree L (2021) "Split-and-Click" sgRNA. in Methods in molecular biology (Clifton, N.J.)

 
Description CRISPR is a remarkable gene editing technology developed by Doudna and Charpentier. We have used our click chemistry methodology to produce libraries of CRISPR/Cas constructs that can be sued to edit multiple genes (or multiple loci in a given gene) simultaneously. We have started to receive queries from research groups who would like to use this methodology.
Exploitation Route The outcomes will be of value to academic and industrial groups working on gene editing for imaging, diagnostics and therapeutics.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology