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September 5th, 2025
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University of Toronto
molecular biology
bioRxiv

Deep-mutational scanning libraries using tiled-region exchange mutagenesis

Kindree, K.Open in Google Scholar•Chochinov, C. A.Open in Google Scholar•Bhachu, K.Open in Google Scholar•Cheng, Y.Open in Google Scholar•Caron, A.Open in Google Scholar•McDonald, M.Open in Google Scholar•Mamai, Z.Open in Google Scholar•Nguyen Ba, A. N.Open in Google Scholar

The analysis of gene function frequently requires the generation of mutants. Deep-mutational scanning (DMS) has emerged as a powerful tool to decipher important functional residues within genes and proteins. However, methods for performing DMS tend to be complex or laborious. Here, we introduce Tiled-Region Exchange (T-REx) Mutagenesis, which is a multiplexed modification of the EMPIRIC mutagenesis approach. Self-encoded removal fragments are cloned in parallel in non-overlapping gene locations and pooled. In a one-pot reaction, oligonucleotides are then swapped with their corresponding self-encoded removal fragments in bulk using a single Golden Gate reaction. To aid in downstream phenotyping, the library is then fused with unique DNA barcodes using the Bxb1 recombinase. We demonstrate this approach and its optimizations, to show that it is both easy to perform and efficient. This method offers simple and expedient means to create comprehensive mutagenesis libraries.

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