Project CRISPR- discovery of regulators in cancer-linked smORF antigen presentation; Reuven Agami

Many small open reading frames (smORFs) in the genome are translated into microproteins that standard protein catalogs miss. Some of the resulting peptides are presented on HLA molecules at the cell surface, where they can act as cancer-specific antigens and potential immunotherapy targets. Yet the molecular machinery that governs which smORF-derived peptides are translated, processed, and presented remains largely uncharted, which limits their therapeutic and diagnostic use.

This project will systematically identify the regulators of smORF-derived cancer antigen presentation using a pooled CRISPR screening approach, built around a novel dual-reporter system that simultaneously monitors smORF translation and HLA surface presentation. Within ORFeus Work Package 3, which investigates the roles of the dark proteome in cancer and immunology, it provides the mechanistic understanding of antigen presentation that underpins the work package's therapeutic pipeline: screen targets are prioritized using candidates from partner projects, and top hits are validated in patient-derived cancer models to connect regulatory insights to clinically relevant settings.

• Develop and deploy a dual-reporter system that simultaneously monitors smORF translation and HLA surface presentation, and establish it as the readout for a pooled CRISPR screen.
• Identify key regulatory factors by screening translation and antigen-processing pathways against targets prioritized from the projects of other doctoral candidates (DCs) in the network, here DC4 on host-pathogen microproteins and DC5 on the secreted microproteome.
• Validate top candidates using immunopeptidomics and test their relevance in patient-derived cancer models together with DC15, aiming for at least one mechanistically characterized regulatory pathway with therapeutic potential.
• Collaborate across the ORFeus network, contribute reporter cell lines and a regulatory map of antigen presentation factors to the shared ORFeome platform, and produce the project's scientific report.

Methods and platforms
: pooled CRISPR screening (including CRISPRa/CRISPRi libraries), dual-reporter cell-line engineering, flow cytometry and cell sorting, immunopeptidomics by mass spectrometry, HLA antigen-presentation assays, and functional validation in patient-derived cancer models, contributing data and cell lines to the ORFeome platform.

Secondment
: you will spend around three months at MyNeo (Ghent, Belgium), applying the project's screening workflows to the identification of personalized cancer antigen candidates. You will also be guided by an independent academic advisor, with the possibility of a short, primarily virtual research exchange to strengthen the project.

MSCA eligibility

You must meet all of the following on your recruitment date:

• You do not already hold a doctoral degree. If you have defended a doctoral thesis but the degree has not yet been formally awarded, you are not eligible.
• Mobility rule: you must not have lived or carried out your main activity (work, studies, and so on) in the Netherlands for more than 12 months in the 36 months immediately before your recruitment date. Compulsory national service, short stays such as holidays, and time spent in a procedure to obtain refugee status under the Geneva Convention do not count toward the 12 months.
• You hold, or will hold before the start date, a degree that formally entitles you to enroll in a doctorate, and you can enroll in the doctoral program at the Netherlands Cancer Institute / Erasmus University Rotterdam.

Candidates of any nationality may apply. There is no limit on prior research experience, as long as you do not already hold a doctorate.

Project-specific profile

• A master's…