Abstract <p>Cyanobacteria are diverse photosynthetic microorganisms of great interest for fundamental science and sustainable biotechnological applications. However, their polyploidy makes genetic manipulation challenging and time-consuming. The development of CRISPR/Cas tools has greatly accelerated genome editing and metabolic engineering of some cyanobacterial model species. In this work, we extend the CRISPR/Cas12a system for targeted gene deletion in the non-model cyanobacterium <i>Cyanothece</i> sp. PCC 7425, interesting for its ability to perform intracellular calcium carbonate (CaCO₃) biomineralization, nitrogen fixation, etc. We demonstrate for the first time its tractability to gene knockout by generating deletion mutants of four genes (<i>cax3-cax4</i>, <i>gor</i>, and <i>sodB</i>) acting in metabolism and/or response to stresses, using Cas12a-mediated homologous recombination. Importantly, full chromosome segregation was rapidly achieved after a single round of selection in all cases. All mutants were genotypically and phenotypically characterised. Moreover, biochemical analysis in the case of the <i>ΔsodB</i> mutant further confirmed its targeted deletion. Overall, CRISPR/Cas12a provides a rapid and efficient system for genome editing in <i>Cyanothece</i> sp. PCC 7425, establishing this organism as a versatile model for studying oxidative stress pathways, metal toxicity, and moreover, the still poorly known mechanism(s) of intracellular CaCO₃ biomineralization.</p> Key points <p>• <i>Rapid and efficient CRISPR/Cas12a editing established in Cyanothece sp. PCC 7425.</i></p> <p>• <i>Fully segregated knockout mutants obtained after a single selection round.</i></p> <p>• <i>Platform for exploring the biotechnological potential of Cyanothece sp. PCC 7425.</i></p>