Pooled tagging of endogenous proteins for exploration of the human proteome driven by prime editing

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Doctor of Philosophy (PhD)
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Bioengineering
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Engineering
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2025
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Sanchez, Henry
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Abstract

Proteome subcellular organization carries important information on protein function and cellular state, which is not reflected in abundance measurements of proteins, RNA or other biomolecules in cells. Endogenous peptide tagging of proteins provides a highly specific handle to directly image and perturb proteins within their native regulatory context, yet such approaches are challenging to scale. In this thesis, we set out to develop a pooled tagging approach driven by prime editing. In Chapter 1, we discuss the background and motivation for developing this pooled tagging approach in the context of past and present work in this emerging area of functional genomics. In Chapter 2, we discuss how we set out to develop this approach and what we found along the way. In Chapter 3, we close with discussion, challenges and future opportunities for pushing this method forward towards genome-wide tagging of endogenous proteins for unbiased exploration of proteome dynamics across cell types and environmental perturbations.

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Shalem, Ophir
Date of degree
2025
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