Design and implementation of CRISPR/Cas9 Prosthetic Gene Networks for Research and Therapeutic applications

Supervisor:  Prof Tudor Fulga

Project Description

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. 

Training

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.

Publications

  1. A multiplexable TALE-based binary expression system for in vivo cell interaction studies. Markus Toegel#, Ghows M. Azzam#, et al. (2017) Nature Comm. In press.
  2. In situ functional dissection of RNA cis-regulatory elements by multiplex CRISPR-Cas9 genome engineering. Qianxin Wu#, Quentin RV. Ferry#, et al. (2017) Nature Comm. In press.
  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.

For further information please contact:  Prof Tudor Fulga