Discovery, characterization, and application of chromosomal integration sites in the hyperthermophilic crenarchaeon Sulfolobus islandicus

Sulfolobus islandicus , an emerging crenarchaeal model organism, offers unique advantages for metabolic engineering and synthetic biology applications owing to its ability to thrive in extreme environments. Although several genetic tools have been established for this organism, the lack of well-characterized chromosomal integration sites has considerably limited its potential as a cellular factory. Here, we systematically identified and characterized 13 artificial CRISPR RNAs (crRNAs) targeting eight chromosomal integration sites in S. islandicus using the CRISPR-COPIES pipeline and a multi-omics-informed computational workflow. By leveraging the endogenous CRISPR-Cas systems, we integrated the reporter gene lacS into these sites and validated heterologous gene expression through a β -galactosidase reporter assay, which revealed significant positional effects on expression levels. As a proof of concept, we utilized these characterized sites to genetically manipulate lipid ether composition by overexpressing GDGT (glycerol dibiphytanyl glycerol tetraether) ring synthase B (GrsB) in S. islandicus , a key enzyme in GDGT biosynthesis. This study expands the genetic toolbox for S. islandicus and highlights a concept that could be widely applicable to other Sulfolobales , advancing their potential as robust platforms for archaeal synthetic biology and industrial biotechnology.