CRISPR-Cas9 Editing Chimerism in Saccharomyces Cerevisiae: Regulation by Cell Cycle and Its Implications for Microbial Metabolic Engineering

Abstract

CRISPR-Cas9 editing often produces chimeric cell populations where edited and unedited cells coexist, undermining yield and reproducibility in industrial microbial processes. Modulation of the cell cycle can reduce chimerism and improve editing fidelity. The review summarizes definitions and detection methods for chimeric populations including flow cytometry and single cell sequencing and highlights representative cases from Bacillus subtilis cellulase production and Escherichia coli isobutanol synthesis that show how partial editing lowers titers and increases byproducts. Evidence is presented that chromatin accessibility and DNA repair pathway choice change across G1, S, G2 and M phases and thereby determine Cas9 cleavage efficiency and repair outcomes. S phase is associated with higher chromatin accessibility and increased homology directed repair activity that favor precise edits. G1 and M phases either favor non homologous end joining or restrict cutting, leading to higher chimerism. Practical strategies such as population synchronization, timed delivery of repair templates and integrated molecular monitoring are evaluated for their potential to reduce population heterogeneity during strain engineering and fermentation. This review aims to clarify the molecular mechanisms by which Saccharomyces cerevisiae cell cycle modulates CRISPR editing chimerism and to provide guidance for optimizing industrial genome engineering.