ArticleG3 (Bethesda, Md.)2026
Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis.
Article in G3 (Bethesda, Md.), 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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8 authors.
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Abstract
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 Extremely Methodical and Parallel Investigation of Randomized Individual Codons mutagenesis approach. Self-encoded removal fragments are cloned in parallel in nonoverlapping gene locations and pooled. In a 1-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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