The expanded CRISPR toolbox for constructing microbial cell factories

Microbial cell factories (MCFs) convert low-cost carbon sources into valuable compounds. The CRISPR/Cas9 system has revolutionized MCF construction as a remarkable genome editing tool with unprecedented programmability. Recently, the CRISPR toolbox has been significantly expanded through the exploration of new CRISPR systems, the engineering of Cas effectors, and the incorporation of other effectors, enabling multi-level regulation and gene editing free of double-strand breaks. This expanded CRISPR toolbox powerfully promotes MCF construction by facilitating pathway construction, enzyme engineering, flux redistribution, and metabolic burden control. In this article, we summarize different CRISPR tool designs and their applications in MCF construction for gene editing, transcriptional regulation, and enzyme modulation. Finally, we also discuss future perspectives for the development and application of the CRISPR toolbox.