Molecular biology techniques like gene editing have altered the specific genes in micro-organisms to increase their efficiency to produce biofuels. This review paper investigates the outcomes of Clustered regularly interspaced short palindromic repeats (CRISPR) for gene editing in extremophilic micro-organisms to produce biofuel. Commercial production of biofuel from lignocellulosic waste is limited due to various constraints. A potential strategy to enhance the capability of extremophiles to produce biofuel is gene-editing via CRISPR-Cas technology. The efficiency of intracellular enzymes like cellulase, hemicellulose in extremophilic bacteria, fungi and microalgae has been increased by alteration of genes associated with enzymatic activity and thermotolerance. extremophilic microbes like Thermococcus kodakarensis, Thermotoga maritima, Thermus thermophilus, Pyrococcus furiosus and Sulfolobus sp. are explored for biofuel production. The conversion of lignocellulosic biomass into biofuels involves pretreatment, hydrolysis and fermentation. The challenges like off-target effect associated with use of extremophiles for biofuel production is also addressed. The appropriate regulations are required to maximize effectiveness while minimizing off-target cleavage, as well as the total biosafety of this technique. The latest discovery of the CRISPR-Cas system should provide a new channel in the creation of microbial biorefineries through site- specific gene editing that might boost the generation of biofuels from extremophiles. Overall, this review study highlights the potential for genome editing methods to improve the potential of extremophiles to produce biofuel, opening the door to more effective and environmentally friendly biofuel production methods.