Project Single-cell translation dynamics targeted smORF editing; Alexander van Oudenaarden

Small open reading frames (smORFs) scattered across the genome are translated into microproteins that standard reference catalogs miss, and these and other elements of the dark proteome can help regulate translation differently from one cell to the next. Most methods read out translation in bulk, averaging over many cells and hiding the cell-to-cell differences that matter during processes such as neural differentiation.

Measuring how a targeted change to a smORF alters translation in individual cells, and doing so at scale, is an open experimental problem at the heart of dark proteome research.

This project will establish an integrated framework that studies translation regulation at single-cell resolution by combining CRISPR-based smORF editing with single-cell ribosome profiling. The novel 'ribo-perturb-seq' approach detects guide RNAs and translation profiles simultaneously in single cells and will be applied to organoid models to investigate translation regulation during neural differentiation. It is a key enabling technology of ORFeus Work Package 2, which moves from the discovery of the dark proteome to understanding its function: the platform provides single-cell functional validation for partner projects on translational control and computational microprotein design, and it connects to partner projects on single-molecule translation mapping and AI-driven translation prediction.

• Adapt Perturb-seq vector designs so that guide RNAs and translation profiles can be detected simultaneously in single cells, establishing the ribo-perturb-seq platform.
• Validate the ribo-perturb-seq approach using smORF variants in various cell lines, generating a quantitative dataset that characterizes how smORF variants affect translation.
• Apply the technology to organoid models to investigate translation regulation during neural differentiation, coordinating experimental designs with the projects of other doctoral candidates (DCs) in the network, here DC7 on lncRNA translation regulation, and drawing on isoform-level translation data from DC2 to inform the single-cell analysis framework.
• Collaborate across the ORFeus network, contribute the ribo-perturb-seq framework and single-cell translation datasets to the shared ORFeome platform, and produce the project's scientific report.

Methods and platforms
: CRISPR-based genome and base editing, Perturb-seq guide RNA libraries, single-cell ribosome profiling (single-cell Ribo-seq) and single-cell RNA sequencing, cell-line and organoid culture including neural differentiation models, and single-cell data analysis, contributing methods and datasets to the ORFeome platform.

Secondment
: you will spend around three months at an ORFeus industry partner, applying cutting-edge single-cell proteomics to investigate translation dynamics. 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 Hubrecht Institute / Utrecht University.
• 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 degree (or equivalent) in molecular biology, cell biology, biotechnology, genetics, biomedical sciences, or a related life-science field.
• Hands-on experimental experience in molecular biology, for example CRISPR-based editing, single-cell techniques, or next-generation sequencing library preparation.
• Desirable: experience with single-cell methods, ribosome profiling, CRISPR screening or base editing, organoid or stem-cell culture, and basic data analysis (for example R or Python), and an interest in microproteins and the dark…