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CRISPR base editor-based targeted random mutagenesis (BE-TRM) toolbox for directed evolution

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Directed evolution (DE) of desired locus by targeted randommutagenesis (TRM) tools is a powerful approach for generatinggenetic variations with novel or improved functions, particularlyin complex genomes. TRM-based DE involves developing amutant library of targeted DNA sequences and screening thevariants for the desired properties. However, DE methods havefor a long time been confined to bacteria and yeasts. Lately,CRISPR/Cas and DNA deaminase-based tools that circumventenduring barriers such as longer life cycle, small library sizes,and low mutation rates have been developed to facilitate DE innative genetic environments of multicellular organisms. Notably,deaminase-based base editing-TRM (BE-TRM) tools havegreatly expanded the scope and efficiency of DE schemes byenabling base substitutions and randomization of targeted DNAsequences. BE-TRM tools provide a robust platform for thecontinuous molecular evolution of desired proteins, metabolicpathway engineering, creation of a mutant library of desiredlocus to evolve novel functions, and other applications, suchas predicting mutants conferring antibiotic resistance. This reviewprovides timely updates on the recent advances in BE-TRMtools for DE, their applications in biology, and future directionsfor further improvements.

Abstract

Directed evolution (DE) of desired locus by targeted randommutagenesis (TRM) tools is a powerful approach for generatinggenetic variations with novel or improved functions, particularlyin complex genomes. TRM-based DE involves developing amutant library of targeted DNA sequences and screening thevariants for the desired properties. However, DE methods havefor a long time been confined to bacteria and yeasts. Lately,CRISPR/Cas and DNA deaminase-based tools that circumventenduring barriers such as longer life cycle, small library sizes,and low mutation rates have been developed to facilitate DE innative genetic environments of multicellular organisms. Notably,deaminase-based base editing-TRM (BE-TRM) tools havegreatly expanded the scope and efficiency of DE schemes byenabling base substitutions and randomization of targeted DNAsequences. BE-TRM tools provide a robust platform for thecontinuous molecular evolution of desired proteins, metabolicpathway engineering, creation of a mutant library of desiredlocus to evolve novel functions, and other applications, suchas predicting mutants conferring antibiotic resistance. This reviewprovides timely updates on the recent advances in BE-TRMtools for DE, their applications in biology, and future directionsfor further improvements.

참고문헌 (59)

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