Engineering aynthetic Circuits for research and therapeutic applications using CRISPR-based sensors and extracellular vesicles

Lead Research Organisation: University of Oxford
Department Name: Synthetic Biology DTC

Abstract

1. Development of CRISPR-based sensors for cellular reprogramming

1.1 Brief introduction
Tracking RNA expression in living cells is essential in understanding development, disease and cellular responses to environmental stimuli. The goal of this study is to develop a new platform for live RNA detection and actuation in mammalian cells using Cas9-based transcriptional-activators coupled with inducible spacer-blocking hairpins single guide RNAs (iSBH-sgRNA) developed by Ferry et al.1
1.2 Specific aims
a. Providing proof-of-concept that iSBH-sgRNA can be used for detection of exogenously expressed target RNAs
b. Understanding base-pairing requirements between synthetic RNA triggers and iSBH-sgRNA
c. Extending length of synthetic triggers and determining the tolerated range of trigger lengths
d. Understanding how RNA secondary structures impact trigger activity
e. Extending the original iSBH-sgRNA design and identifying the optimal configurations for detection of long RNA triggers
f. Development of a computational pipeline for screening sub-sequence domains in endogenous targets that could act as triggers. Starting from endogenous RNA targets, this pipeline would output an optimal sequence for iSBH-sgRNAs
g. Test and optimise computational pipeline using bacterial systems. Link detection of target mRNAs with transgene expression
h. Test and optimise computational pipeline in mammalian cells
i. Develop disease-oriented applications by linking detection of endogenous target RNAs with expression of therapeutic transgenes or endogenous genes

2. Development of strategies for loading and delivering CRISPR components using extracellular vesicles

2.1 Brief introduction
Due to their non-immunogenic properties and their ability to cross the blood-brain barrier, extracellular vesicles (EVs) have become valuable tools for drug delivery as well as for engineering communication between mammalian cells3. Although recent studies have attempted to deliver Cas9 to target cells using EVs, limited success has been achieved so far 4,5. This project aims to establish more efficient methods for Cas9 delivery using EVs and to developed entailing EV-based delivery of programable transcriptional activators of clinically relevant genes.
2.2 Specific aims
a. Providing proof-of-concept evidence that EVs could be loaded with Cas9 transcriptional activators and guide RNAs
b. Developing reporter cell lines that trigger expression of fluorescent markers in the presence of active Cas9 transcriptional activators
c. Providing proof-of-concept evidence that EVs loaded with Cas9 transcriptional activators could trigger detectable responses in target cells
d. Performing a mouse biodistribution study for understanding which cells might represent good targets for the modified EVs
e. Developing this system towards upregulation of clinically-relevant gene targets

3. Engineering behaviour of target cellular populations using CRISPR-based sensors and extracellular vesicles

This third project will aim to combine the technologies developed in Part 1 and Part 2
3.1 Project conditions
- Successful implementation of inducible gRNAs capable of linking detection of disease-associated RNAs with the expression of therapeutically relevant genes
- Successful delivery of Cas9-transcriptional activators to target cells using EVs

3.2 Specific aims
a. Proof-of-principle evidence that inducible gRNAs could restore disease phenotype in vitro
b. Loading Cas9-transcriptional activators and iSBH-sgRNA into EVs
c. Optimising the amount of Cas9 and iSBH-sgRNA to be delivered to target cells for obtaining a relevant cellular response
d. Testing strategy in mouse models

This project falls within the EPSRC Synthetic Biology research area and will include EvOX Therapeutics as an industrial partner.

Publications

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Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/R512333/1 01/10/2017 30/09/2021
1935000 Studentship EP/R512333/1 01/10/2017 30/09/2021 Oana Pelea