Tools for engineered biology

1. A robust pipeline for the production and screening of synthetic transcription factors

Lead: Professor Steven Pollard

Aim: To design, build and test a set of novel synthetic transcription factors (sTFs) based upon the TALE and dCas9 architectures. The initial goal is to identify those sTFs that are capable of activating ‘master regulator’ endogenous target genes. These will then be deployed in combinations to create the desired levels and combinations of reprogramming factors to enable efficient conversion of mammalian cell types.

Outputs:

A bespoke DNA assembly method for mammalian systems was developed and is described in the following paper. 

Martella, A., et al.  (2017) ‘EMMA: An Extensible Mammalian Modular Assembly Toolkit for the Rapid Design and Production of Diverse Expression Vectors.’ ACS Synthetic Biology, 6(7) pp. 1380 - 1392

2. Synthetic transcription factors for tuneable activation of endogenous mammalian master regulators

Lead: Dr Abdenour Soufi

Aim: Pioneer transcription factors have the ability to reprogram silent chromatin. Pioneer TFs of interest are Oct 4, Sox2, Klf4 (OSK – all pioneer TFs) and c-Myc, which together can reprogramme fibroblasts to becoming induced pluripotent stem cells (iPSCs). Our aim is to define functionally minimum domains for engineering synthetic pioneer TFs and use them for robust cellular reprogramming.

Outputs: A paper is currently in press

3. A toolkit for small-molecule control of engineered proteins

Lead: Professor Jamie Davies

Aim: To create a database and interface to facilitate the design of drugability into proteins, and to provide proof of concept that it is feasible to build a druggable-unit into a protein previously not responsive to that drug.

Outputs:

• The database is available here http://synpharm.guidetopharmacology.org/  and outined in Ireland SM et al. (2018) - see below 
• We have completed the design, construction and testing of versions of both Cas9 and Cpf1 CRISPR nucleases that have novel drug control engineered into them. 

• The work has also informed new optogenetic studies with collaborators in Germany (Matias Zurbriggen, Düsseldorf)   

Read more here: 

Ireland SM et al. (2018) SynPharm, a Guide to PHARMACOLOGY database tool for designing drug control into engineered proteins. ACS Omega, 3(7):7993-8002. doi: 10.1021/acsomega.8b00659

Dominguez-Monedero A, Davies JA (2018) Tamoxifen- and mifepristone- inducible versions of CRISPR effectors, Cas9 and Cpf1. ACS Synthetic Biology, 7(9):2160-2169. doi: 10.1021/acssynbio.8b00145

Baaske J, et al(2018)  Dual-controlled optogenetic system for the rapid down-regulation of protein levels in mammalian cells. Sci Rep 8(1):15024. doi: 10.1038/s41598-018-32929-7

4. Construction of human synthetic chromosomes

Aim: To find combinations of chromatin modifiers that increase the efficiency of synthetic centromere formation in cell lines of clinical and experimental utility. We will explore the use of synthetic chromosomes in high throughput screens for compounds that induce aneuploidy. Such compounds are potential agents for chemotherapy, as many tumour cells appear to tolerate the induction of aneuploidy less than non-transformed cells.

Lead: Professor Wiliam Earnshaw

Outputs:

• We have fully characterised a novel type of HAC with centrochromatin and heterochromatin domains (named ^hybridHAC); this has been used as a platform to do double tethering experiments and to study the interaction of proteins with opposing effects on centromere stability. This has informed the development of more sophisticated HACs for the future.

Read more here: 

Pesenti E, et al. (2018) Generation of a Synthetic Human Chromosome with Two Centromeric Domains for Advanced Epigenetic Engineering Studies, ACS Synth. Biol., 7 (4), pp 1116–1130 DOI: 10.1021/acssynbio.8b00018

Kouprina N, et al (2018) Human Artificial Chromosome with Regulated Centromere: A Tool for Genome and Cancer Studies ACS Synth. Biol., 7 (9), pp 1974–1989 DOI: 10.1021/acssynbio.8b0023

5. Development of chromosomal synthetic safe-harbour landing pads

Aim: To build circuits of increasing complexity requires the introduction of ever-larger numbers of genetic constructs, and transient transfection methods are unreliable and impractical. To address this problem, we have developed a novel ‘landing pad’ that can be integrated as a transposon into the genome of the desired cell type.

Lead: Professor Susan Rosser

Outputs: We are developing a suite of ‘landing pads’ that can be integrated as a transposon into the genome of the desired cell type. Our first prototype landing pad contained three independent recombinase attachment sites that can each be used for integrase-mediated integration or cassette exchange.

Publications pending.