Improving

Bacillus subtilis is the model Gram-positive and industrial chassis bacterium; it has blossomed as a robust and promising host for enzyme, biochemical, or bioflocculant production. However, synthetic biology and metabolic engineering technologies of B. subtilis have lagged behind the most widely used industrial chassis Saccharomyces cerevisiae and Escherichia coli. CRISPR (an acronym for clustered regularly interspaced short palindromic repeats) enables efficient, site-specific, and programmable DNA cleavage, which has revolutionized the manner of genome editing. In 2016, CRISPR technology was first introduced into B. subtilis and has been intensely upgraded since then. In this Review, we discuss recently developed key additions to CRISPR toolkit design in B. subtilis with gene editing, transcriptional regulation, and enzyme modulation. Second, advances in the B. subtilis chassis of efficient biochemicals and proteins with CRISPR engineering are discussed. Finally, we conclude with perspectives on the challenges and opportunities of CRISPR-based biotechnology in B. subtilis, wishing that B. subtilis can be comparable to traditional industrial microorganisms such as E. coli and S. cerevisiae someday soon.