Streamlined and efficient genome editing inCupriavidus necatorH16 using an optimised SIBR-Cas system

Cupriavidus necator H16 is a promising microbial platform strain for CO 2 valorisation. While C. necator is amenable to genome editing, existing tools are often inefficient or rely on lengthy protocols, hindering its rapid transition to industrial applications. In this study, we simplified and accelerated the genome editing pipeline for C. necator by harnessing the Self-splicing Intron-Based Riboswitch (SIBR) system. We used SIBR to tightly control and delay Cas9-based counterselection, achieving >80% editing efficiency at two genomic loci within 48 hours after electroporation. To further increase the versatility of the genome editing toolbox, we upgraded SIBR to SIBR2.0 and used it to regulate the expression of Cas12a. SIBR2.0-Cas12a could mediate gene deletion in C. necator with ∼70% editing efficiency. Overall, we streamlined the genome editing pipeline for C. necator , facilitating its potential role in the transition to a bio-based economy.