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

Area of Science:

• Molecular Biology
• Biotechnology
• Genetics

Background:

• Directed evolution (DE) is crucial for generating genetic variations with improved functions.
• Traditional DE methods were limited to simpler organisms like bacteria and yeasts.
• Barriers such as long life cycles and low mutation rates hindered DE in multicellular organisms.

Purpose of the Study:

• To review recent advances in base editing-TRM (BE-TRM) tools for DE.
• To highlight the expanded scope and efficiency of BE-TRM in multicellular systems.
• To discuss applications and future directions of BE-TRM in biological research.

Main Methods:

• CRISPR/Cas and DNA deaminase-based tools have been developed to overcome DE limitations.
• Base editing-TRM (BE-TRM) enables targeted base substitutions and sequence randomization.
• Creation and screening of mutant libraries are central to the DE process.

Main Results:

• BE-TRM tools significantly enhance the scope and efficiency of DE schemes.
• These tools facilitate DE in the native genetic environments of multicellular organisms.
• BE-TRM supports continuous molecular evolution for protein engineering and metabolic pathway optimization.

Conclusions:

• BE-TRM represents a significant advancement in DE methodologies.
• It offers a robust platform for engineering novel functions and predicting traits like antibiotic resistance.
• Further improvements in BE-TRM tools will continue to expand their biological applications.