Technical limitations of CRISPR-Cas9 genome editing in bacteria: challenges and future directions.

The CRISPR-Cas system, which originated as an adaptive immune system in bacteria and archaea, has been repurposed as a precise and programmable tool for genetic manipulation in both prokaryotes and eukaryotes. Its applications in bacteria include targeted genome modifications, antimicrobial resistance studies, functional genomics studies, and the development of engineered strains for industrial and synthetic biology applications. Therefore, the understanding of technical aspects of CRISPR systems and their underlying molecular mechanisms is essential for experimental accuracy, reproducibility, and biosafety. CRISPR editing introduces several challenges in bacteria, including off-target effects, DNA repair limitations, cytotoxicity of Cas nucleases, and host-specific restriction-modification barriers. In addition to these specific challenges, metabolic burden, sgRNA design, and delivery challenges further introduce limitations. Such issues compromise editing efficiency, genomic stability, and cell viability. Recent studies have focused on improved guide RNA design, alternative Cas variants, refined delivery strategies, and host-adapted engineering as promising directions to enhance editing. This review discusses the principal barriers to CRISPR-Cas9 genome editing of bacteria, evaluates the current strategies for addressing these barriers, and highlights emerging approaches aimed at improving the efficiency, reliability and precision in bacterial genome engineering.