# Deep-mutational scanning libraries using Tiled-Region Exchange mutagenesis

## TL;DR

This paper introduces a new method called T-REx mutagenesis to simplify and speed up the creation of gene mutant libraries for studying gene function.

## Contribution

The novel T-REx mutagenesis method enables efficient and parallel generation of deep-mutational scanning libraries.

## Key findings

- T-REx mutagenesis allows for the parallel cloning of self-encoded removal fragments in nonoverlapping gene regions.
- A single Golden Gate reaction enables bulk oligonucleotide swapping with removal fragments.
- The method is efficient and easy to perform, as demonstrated through optimizations and practical implementation.

## 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.

Graphical AbstractFor image description, please refer to the figure legend and surrounding text.