CRISPR/Cas9 technology is one of the common methods of genome editing and targeted gene mutation, which has recently been used for manipulating microalgae such as Chlamydomonas reinhardtii. Besides, this technology can play a role in the fight against greenhouse gases (e.g., carbon dioxide) production by studying genetic pathways to improve algal strains. Among several genes in algae that respond to CO₂ and regulators control the expression of each; Cia5 is one of the most critical transcriptional regulators. In this research,we knocked out the Cia5 gene using the CRISPR/Cas9 technique and analysed the ability of C. reinhardtii to perform CO₂ sequestration. Our results showed that C. reinhardtii has better performance (i.e., response to CO₂ treatment) in both control and mutant species at 0.5% CO₂ concentration than other concentrations. However, the difference between the control microalgae species and the mutant species was in the CO₂ removal efficiency. Additionally, our findings revealed that the control type isolate in CO₂ concentrations of 0.04%, 0.5% and 1% had removal efficiencies of 27%, 37% and 21%, respectively. Nevertheless, for mutant species in the same concentrations, the observedremoval efficiencies were 16%, 23% and 9%.