Abstract
Abstract
The development of synthetic biology has brought new opportunities for green manufacturing. A growing number of non-model bacteria are increasingly harnessed as chassis organisms based on their unique physiological features. The thermophilic microorganism
Thermus antranikianii
holds great potential as a next-generation cell factory, yet its exploitation is constrained by the lack of genetic engineering tools. Here we report a thermostable CRISPR-BrCas12b-based gene editing tool for this thermophile. The BrCas12b mediates precise target recognition and cleavage at 45–70°C, matching the growth temperature of
T. antranikianii
. With a TTN PAM, BrCas12b provides a wide array of editable sites throughout the
Thermus
genome. This tool facilitates convenient gene inactivation, knockouts, and insertions in the
T. antranikianii
genome, thereby serving as an effective platform for genetic circuit construction and reprogramming, and further expanding the repertoire of gene editing tools for thermophiles.