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

Kortni Kindree1, Claire A Chochinov1, Keerath Bhachu1

  • 1Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario M5S 3G5, Canada.

G3 (Bethesda, Md.)
|January 14, 2026
PubMed
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We developed Tiled-Region Exchange (T-REx) Mutagenesis, a simpler method for creating gene mutation libraries. This deep-mutational scanning technique efficiently identifies key functional residues in genes and proteins.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Gene function analysis often necessitates mutant generation.
  • Deep-mutational scanning (DMS) is crucial for identifying functional residues.
  • Existing DMS methods can be complex and time-consuming.

Purpose of the Study:

  • To introduce Tiled-Region Exchange (T-REx) Mutagenesis, a streamlined approach for creating mutagenesis libraries.
  • To demonstrate the efficiency and ease of use of the T-REx method.
  • To provide a simpler alternative for comprehensive mutagenesis library construction.

Main Methods:

  • T-REx Mutagenesis modifies the EMPIRIC approach using multiplexed oligonucleotide swapping.
  • Self-encoded removal fragments are cloned in parallel and pooled.
Keywords:
cloningdeep-mutational scanning

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  • A single Golden Gate reaction facilitates bulk oligonucleotide exchange.
  • Bxb1 recombinase is used to fuse unique DNA barcodes for phenotyping.

    • Main Results:

    • The T-REx Mutagenesis method was successfully implemented and optimized.
    • The approach was shown to be both easy to perform and highly efficient.
    • Comprehensive mutagenesis libraries can be created using this technique.

    Conclusions:

    • Tiled-Region Exchange (T-REx) Mutagenesis offers a significant advancement in creating gene mutation libraries.
    • This method simplifies the process of deep-mutational scanning.
    • T-REx provides an expedient tool for researchers studying gene and protein function.