CUBIC: A Versatile Cumate-Based Inducible CRISPRi System in .

, a genus of Gram-positive bacteria, is known as nature's medicine maker, producing a plethora of natural products that have huge benefits for human health, agriculture, and biotechnology. To take full advantage of this treasure trove of bioactive molecules, better genetic tools are required for the genetic engineering and synthetic biology of . We therefore developed CUBIC, a novel CUmate-Based Inducible CRISPR interference (CRISPRi) system that allows highly efficient and inducible gene knockdown in . Its broad application is shown by the specific and nondisruptive knockdown of genes involved in growth, development and antibiotic production in various species. To facilitate hyper-efficient plasmid construction, we adapted the Golden Gate assembly to achieve 100% cloning efficiency of the protospacers. We expect that the versatile plug-and-play CUBIC system will create new opportunities for research and innovation in the field of .