CRISPR-Cas Tools for Dissecting and Engineering Environmental Responses in Microalgae.

Microalgae are photosynthetic eukaryotes that need to maintain productivity under variable light, temperature, nutrient and CO2 conditions, yet their complex regulatory networks have made relevant engineering difficult. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated proteins (Cas) have changed the situation, shifting microalgal research from random transgene integration toward precise editing of the genes that mediate photosynthesis, stress responses and environmental acclimation. This work reviews how CRISPR-Cas has been applied across microalgal lineages to (i) dissect genes involved in stress response and photosynthesis, (ii) streamline genomes through large-scale deletions, (iii) build stress-tolerant chassis that retain photosynthetic performance under unsatisfactory conditions and (iv) reprogramme lipid, pigment and high-value compound pathways. We then discuss the determinants of editing efficiency, including single-guide RNA design, Cas nuclease choice and engineering, species-specific delivery, host physiology (cell-wall architecture, cell cycle phase and stress-induced repair-pathway shifts) as well as screening workflows using endogenous markers and high-throughput phenotyping. We propose that integrating species-specific multi-omics analyses, artificial intelligence-assisted design and precise and controllable editing strategies will further enhance the ability of CRISPR-Cas systems to elucidate photosynthetic regulatory networks and environmental response mechanisms in microalgae, while facilitating the optimisation of microalgal metabolic traits and the engineering of environmental adaptability.