CRISPR–Cas9 genome editing in microorganisms: Principles, tools, and applications

CRISPR–Cas9 genome editing has emerged as one of the most transformative technologies in modern molecular biology, fundamentally reshaping genetic manipulation in microorganisms. Derived from an adaptive immune system in prokaryotes, CRISPR–Cas9 enables precise, efficient, and programmable modification of DNA sequences. Its simplicity, scalability, and adaptability have accelerated advances in microbial genetics, metabolic engineering, synthetic biology, and biotechnology. In microorganisms, CRISPR–Cas9 has been widely applied to gene knockout, gene insertion, transcriptional regulation, and pathway optimization. These capabilities have facilitated the development of improved microbial strains for industrial fermentation, biofuel production, pharmaceutical synthesis, agricultural biocontrol, and environmental remediation. Despite its immense potential, the application of CRISPR–Cas9 in microorganisms faces challenges such as off-target effects, delivery efficiency, cytotoxicity, and regulatory constraints. Ongoing innovations, including CRISPR variants, base editing, prime editing, and CRISPR interference systems, continue to expand the versatility and safety of this technology. This review provides a comprehensive overview of CRISPR–Cas9 principles, molecular tools, and editing strategies in microorganisms. Furthermore, it discusses key applications across industrial, agricultural, and environmental sectors, highlighting recent advances and future prospects. By synthesizing current knowledge, this review aims to support researchers in effectively applying CRISPR–Cas9 systems to microbial biotechnology and to guide future developments toward more precise, sustainable, and ethically responsible genome engineering.