CRISPR Screen Multiplexing for Uncharacterised Region Function (SMURF)
Dr L Prendergast
Dr M Martin
Prof S Wedge
No more applications being accepted
Funded PhD Project (European/UK Students Only)
CRISPR-Cas9 screening utilising tiling-sgRNA design across genes, permits functional evaluation of protein domains and precise mapping of critical amino acids. When Indels result in frameshifts or stop codons, protein function is lost, however mutation can also drive gain of protein function. CRISPR cannot yet predict what biologically critical protein domains are amenable to small molecule intervention. Similarly, small molecule drug discovery screening libraries (e.g FragLite) cannot accurately predict which druggable sites, if targeted on a protein, will have maximum biological impact.
Our project combines both approaches to identify functionally important proteins domains, specifically amenable to small molecule intervention.
By exploiting cutting edge molecular methods to directly edit cancer cell genomes, we will investigate unknown relationships between protein structure and function.
CRISPR-Cas9 coupled to sgRNAs efficiently generates DSBs or DNA nicks across the genome. When DSBs are repaired by non-homologous end joining (NHEJ), a cellular DNA damage repair pathway, Indels are formed. Indels are inserions or deletions in the endogenonus DNA sequence. Each gRNA coupled to Cas9 can generate a specific perturbation area or ‘footprint’ of Indels in and around the Cas9 cut site. When multiple gRNAs are overlapped across a gene or target genomic region, this is called CRISPR tiling. Tiling can be used to interrogate whether a region of gene (corresponding to a target protein) is essential for protein function and ultimately cell survival.
Recent bioinformatics advances have deconvolved data from tiling screens to predict functional regions across the genome (Hsu, J.Y et al. Nat Methods (2018)). This confirmed that the choice of Cas9 used in tiling can greatly influence the resolution of the tiling screens, with CRISPRi/CRISPRa providing a ‘broad strokes’ approach (200nt resolution) and Cas9 nucleases providing 7-10nt resolution, allowing more precise mapping of key amino acids.
FragLite is a platform deveolped at Newcastle University to identfy druggable sites on target proteins (Wood, DJ et al. J Med Chem (2019)). In this project, we aim to examine FragLite identified small molecule binding sites, with targeted CRISPR tiling to discover new critical domains for protein function. The project will yield outcomes in the areas of drug discovery, disease and fundamental biology:
• We will use CRISPR tiling, combined with cellbased assays, to interrogate the functional importance of protein pockets identified by FragLite (or other approaches) on a target-bytarget basis.
• We envisage our approach will enable de novo interrogation of protein domain function, for example the identification and quantification of protein:protein interactions.
• Ultimately, Genome wide approaches (ChIP Seq: RNAseq) on CRISPR-tiled cell lines will probe how gain-of-function mutations affect both the epigenetic landscape and regulation of transcription in human tumour cell lines.
Our research plan has two main phases:
Phase 1: CRISPR tiling validation
CDK2 is a well-characterised cell cycle protein with known protein binding partners. Previous CDK2 FragLite studies have led to the identification of a number of ligandable binding sites. These binding sites on CDK2 will be used as target sites for high resolution tiling gRNA design. This will validate our approach and allow refinement of the tiling approaches including Cas9 variant selection.
Phase 2: CRISPR tiling implementation
We ultimately aim to use SMURF on one or more novel targets to allow us to validate the approach as part of the drug discovery bioscience pipelines that support the identification of druggable targets.
Genomic and proteomic approaches (NGS, immunoprecipitation, mass spectrometry) will allow us to interrogate changes in the target genome and proteome. In particular, genome-wide and amplicon sequencing of tiled cell lines with gain-of-function mutations will identify known and novel epigenetic changes associated with target activation, similar to disease states where overexpression occurs.
Implementation of the combined screen approach will establish the basis of this technology for use in drug discovery. We will determine whether CRISPR tiling can reveal new functional domains in proteins, and whether tiling induced mutations can be used to understand how key mutations may underlie structural changes associated with drug resistance.
The Cancer Research UK Newcastle Drug Discovery Unit is an integrated team of cancer biologists, pharmacologists, structural biologists and medicinal chemists who develop novel, differentiated, small molecule therapies for cancer patients.
Assembling experience from industry and academia, we specialise in structural and biophysical fragment-based approaches for drug discovery complemented by our strengths in synthetic chemistry.
We also aim to develop innovative cutting-edge technologies that enable us to better validate targets, assess target ligandability and identify chemical hit matter.
Newcastle drug discovery projects have contributed to the discovery of two registered cancer medicines and two other drugs currently under evaluation in clinical trials
Training & Skills
The sucessful student will be based at the CRUK Newcastle Drug Discovery Unit (DDU) at Newcastle University. As part of the Centre for Doctoral Training, the project will also involve interactions with industry partners.
The experiments proposed in this study bring together expertise in the fields of gene editing, molecular biology, chemistry and structural biology.
We have designed our proposal around an innovative training programme which, in addition to providing comprehensive training, will also allow the student to benefit from unique interdisciplinary expertise across the Drug Discovery Bioscience, Strucutural biology and Medicinal Chemistry teams, obtaining training in advanced research skills including molecular, protein and mammalian cell biology.
Dr. Lisa Prendergast
[Email Address Removed]
How to Apply
You must apply through the University’s online postgraduate application system
You will need to:
• Insert the programme code 8207F in the programme of study section
• Select ‘PhD in Molecular Sciences’ as the programme of study
• Please include the studentship reference code that you are applying for in the studentship/partnership reference field (codes are outlined on the individual projects found on the MoSMed website: research.ncl.ac.uk/mosmed/)
• Attach a CV and cover letter. The cover letter must state the title of the studentship, quote the relevant reference code and state how your interests and experience relate to the project
• Attach degree transcripts and certificates and, if English is not your first language, a copy of your English language qualifications
• Email a copy of your CV and cover letter to: [Email Address Removed] confirming the project that you have applied for