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Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution

  • Rahul Mahadev Shelake (Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University) ;
  • Dibyajyoti Pramanik (Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University) ;
  • Jae-Yean Kim (Division of Applied Life Science (BK21 Four Program), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University)
  • Received : 2023.05.22
  • Accepted : 2023.08.16
  • Published : 2024.01.31
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Abstract

Directed evolution (DE) of desired locus by targeted random mutagenesis (TRM) tools is a powerful approach for generating genetic variations with novel or improved functions, particularly in complex genomes. TRM-based DE involves developing a mutant library of targeted DNA sequences and screening the variants for the desired properties. However, DE methods have for a long time been confined to bacteria and yeasts. Lately, CRISPR/Cas and DNA deaminase-based tools that circumvent enduring barriers such as longer life cycle, small library sizes, and low mutation rates have been developed to facilitate DE in native genetic environments of multicellular organisms. Notably, deaminase-based base editing-TRM (BE-TRM) tools have greatly expanded the scope and efficiency of DE schemes by enabling base substitutions and randomization of targeted DNA sequences. BE-TRM tools provide a robust platform for the continuous molecular evolution of desired proteins, metabolic pathway engineering, creation of a mutant library of desired locus to evolve novel functions, and other applications, such as predicting mutants conferring antibiotic resistance. This review provides timely updates on the recent advances in BE-TRM tools for DE, their applications in biology, and future directions for further improvements.

Keywords

Acknowledgement

This work was supported by the National Research Foundation of Korea (grants NRF 2021R1I1A3057067, 2021R1A5A8029490, 2022R1A2C3010331) and the Program for New Plant Breeding Techniques (NBT, PJ01686702), Rural Development Administration (RDA), Korea.

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