Design and Implementation of CRISPR/Cas9-Based Prosthetic Gene Networks for Research and Therapeutic Applications
The advent and evolution of CRISPR-Cas9 RNA-guided endonucleases has revolutionised the field of genome engineering. The system consists of two fundamental components: a Cas9 nuclease capable of producing DNA double strand breaks and a single guide RNA (sgRNA) required for targeting Cas9 to any given N20NGG DNA sequence by reprogramming its spacer sequence (first 20 nucleotides at the 5’ end). The simplicity of this system is undoubtedly one of the key advantages over pioneering site-directed nucleases (ZFNs and TALENs), which require protein engineering or complex cloning approaches for altering their target specificity.
Due to the effortless design, highly efficient DNA cleavage activity and continuous improvements aimed to increase on-target efficiency and reduce off-target effects, the CRISPR-Cas9 system constitutes an optimal platform for genome engineering-based therapeutic applications. Furthermore, the Cas9 nuclease has been recently repurposed to create programmable transcription regulators in mammalian cells. Consequently, the output expression of any gene of interest can now be controlled by tethering various effector domains to the sgRNA-dCas9 complex. The overarching goal of this project will be to expand the potential of CRISPR-based RNA-guided genome engineering in order to enable the generation of programmable gene networks responsive to exogenous cues and/or endogenous metabolites. Achieving spatial-temporal control over endogenous gene expression will be instrumental for understanding gene pathway architectures, as well as changes and choices in transcriptional programs operating during differentiation, development and disease. Adding to a growing toolkit of standardised components, this conceptual framework will play a pivotal role in synthetic biology, empowering scientists to create complex devices able to challenge or reprogram cells upon sensing a disease state.
The proposed project will be undertaken at University of Oxford’s MRC Weatherall Institute of Molecular Medicine, in a highly dynamic and competitive environment. The project will involve a broad range of cutting edge technologies including CRISPR-Cas9 genome engineering, digital PCR, next generation sequencing, advanced molecular biology, RNA biochemistry, FACS, lentiviral-mediated delivery, and computational biology. In addition, the student will be trained to develop writing and presentation skills, will have opportunities to present her/his work at international conferences and attend at least one advanced training course.
As well as the specific training detailed above, students will have access to a wide-range of seminars and training opportunities through the many research institutes and centres based in Oxford. Students are also able to attend the Methods and Techniques course run by the MRC Weatherall Institute of Molecular Medicine. This course runs through the year, ensuring that students have the opportunity to build a broad-based understanding of differing research techniques.
Generic skills training is offered through the Medical Sciences Division's Skills Training Programme. This programme offers a comprehensive range of courses covering many important areas of researcher development: knowledge and intellectual abilities, personal effectiveness, research governance and organisation, and engagement, influence and impact. Students are actively encouraged to take advantage of the training opportunities available to them.
The department has a successful mentoring scheme, open to graduate students, which provides an additional possible channel for personal and professional development outside the regular supervisory framework. We hold an Athena SWAN Silver Award in recognition of our efforts to support the careers of female students and staff.
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|3||Engineering synthetic signalling pathways with programmable dCas9-based chimeric receptors. Toni A. Baeumler, Ahmed A. Ahmed and Tudor A. Fulga. (2017) Cell Reports. Sep 12;20(11):2639-2653.|
|4||Rational design of inducible CRISPR guide RNAs for de novo assembly of transcriptional programs. Quentin R. Ferry, Radostina Lyutova and Tudor A. Fulga. (2017) Nature Comm. Mar 3;8:14633.|