The CRISPR technology: A promising strategy for improving dark fermentative biohydrogen production using Clostridium spp.

Generating hydrogen gas (H 2) using the dark fermentation method has attracted much attention due to its lower energy requirement and environmental friendliness. However, producing a high yield of bio-H 2 is as challenging as ever due to low energy conversion by microorganisms. In this respect, the advancement of genome editing tools including the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas technology could overcome the established maximum ceiling of product yield. To date, CRISPR-Cas systems, particularly those based on Type II CRISPR-Cas9 and Type V CRISPR-Cas12, are widely used in manipulating novel bacteria to improve the yield of specific biofuel. However, studies using the CRISPR-Cas technology for improving bio-H 2 production remain scarce. Understanding the metabolic pathways of Clostridium spp. is essential for using the CRISPR-Cas technology Thus, this review highlighted the state-of-the-art in CRISPR-Cas systems for bacterial genome editing while paying attention to bioprocess optimization strategies for modulating the biohydrogen production. • Cas9, Cas12 and endogenous CRISPR-Cas can be used for genome editing in Clostridium. • Template vectors for easy deliveries of CRISPR-Cas components in Clostridium spp. • Overexpress butyrate and knockout acetate production pathway improve H 2 production. • Simultaneous repression of solvent and lactate pathway for efficient H 2 production. • Modulation of lignocellulose and sugar utilization genes to improve H 2 production. [ABSTRACT FROM AUTHOR]