Coupling hfCas13d with Circular gRNA Array Precursors Extends RNA Knockdown and Improves Biocatalysis in Yeast

CRISPR-based RNA cleavage holds promise for gene knockdown but its applications in metabolic engineering remain largely unexplored. Here we reconstitute three mammalian CRISPR-based RNA cleavage systems to arrest the intermediate metabolic reactions in the yeast Candida viswanathii to produce hydroxydecanoic acid (HDA), an important nylon precursor. We identify hfCas13d as the optimal effector and next develop a circular guide RNA (CgRNA) precursor architecture to enhance and extend the knockdown of genes involved in HDA production. This hfCas13d/CgRNA precursor platform also represses genes in other yeasts and E. coli, demonstrating broad microbial applicability. Single and dual CgRNA precursor arrays enable multiplexed pathway repression, with dual arrays reducing byproduct formation and increasing HDA titer to 17.8 g/L in shake flasks. Bioreactor fermentation further boosts HDA production to 37.2 g/L. Together, this work establishes hfCas13d/CgRNA as a programmable RNA regulatory platform for engineering microbial cell factories. CRISPR-based RNA cleavage holds promise for gene knockdown but its applications in metabolic engineering remain largely unexplored. Here the authors develop a circular guide RNA architecture to enhance the knockdown of genes involved in HDA production.