Deep-mutational scanning libraries using tiled-region exchange mutagenesis

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.

基因功能研究通常需要构建突变体。深度突变扫描（Deep-mutational scanning, DMS）现已成为解析基因与蛋白质关键功能残基的高效工具。然而，现有DMS实施方法往往存在操作复杂、耗时费力的问题。本文提出平铺区域交换（Tiled-Region Exchange, T-REx）诱变技术，该技术是对EMPIRIC诱变方法的多重改良版本。研究人员将自编码移除片段在非重叠基因位点并行克隆并混合；随后通过单次Golden Gate反应，在单管体系中批量完成寡核苷酸与对应自编码移除片段的替换。为便于下游表型分析，研究人员利用Bxb1重组酶将构建的突变文库与独特DNA条形码（DNA barcode）进行融合。我们对该方法及其优化方案进行了验证，结果表明其不仅操作简便，且效率优异。本方法为构建全面的诱变文库提供了简便快捷的可行途径。