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Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

ABSTRACT: A engineering Pol η variants offers the exciting prospect of further tailoring its activity for base editing. Improving its processivity could ensure that Pol η completes the bypass of the AP site and perhaps extends a few more bases before dissociating, thus preventing the recruitment of other, potentially less desirable, cellular polymerases or repair factors. Base on this hypothesis, We decide to develop a novel T-to-A base editor.

ORGANISM(S): Homo sapiens

PROVIDER: GSE301258 | GEO | 2025/07/08

REPOSITORIES: GEO

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Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

Project description:Developing a T>A Base Editor via Protein Universe Mining and optimizing translesion polymerase

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Project description:Proteins are key molecular players in a cell, and their abundance is extensively regulated not just at the level of gene expression but also post-transcriptionally. Here, we describe a genetic screen in yeast that enables systematic characterization of how protein abundance regulation is encoded in the genome. The screen combines a CRISPR/Cas9 base editor to introduce point mutations with fluorescent tagging of endogenous proteins to facilitate a flow-cytometric readout. We first benchmarked base editor performance in yeast with individual gRNAs as well as in positive and negative selection screens. We then examined the effects of 16,452 genetic perturbations on the abundance of eleven proteins representing a variety of cellular functions. We uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Overall, our novel screening approach provides unprecedented insights into the components, scale and connectedness of the protein regulatory network.

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