In response to the energy crisis microalgae are a promising feedstock for biofuel production. The use of metabolic engineering to improve yields of biofuel-related lipid components in microalgae, without affecting cell growth, is now recognized as a promising and more economically feasible approach to develop more sustainable energy sources. For this, we generated Chlamydomonas mutant strains using CRISPR-Cas9 technology to knockout a gene involved in fatty acid (FA) degradation. In the knockout mutant, total lipid accumulated up to 28% of dried biomass, while that of wild-type (WT) was 22%. This increase was also accompanied by a noticeable shift in FA composition with an increase up to 27.2% in the C18:1 proportion. In addition, these mutants showed comparable growth rate to the WT, indicating that inhibiting lipid catabolism through gene editing technology is a promising strategy to develop microalgal strains for biofuel production.