CRISPR-Cas at a crossroads: from microbial immunity to precision biotechnology.

Abstract

Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) form RNA-guided adaptive immune systems in bacteria and archaea that mediate sequence-specific defense against invading genetic elements. Beyond their ecological role in restricting bacteriophage infection and horizontal gene transfer (HGT), CRISPR-Cas systems have been repurposed as programmable nucleases, enabling rapid, scalable, and precise genome engineering. Over the past decade, CRISPR platforms, most prominently Cas9, have transformed functional genomics, accelerated target discovery and drug development, and progressed from experimental tools to clinically evaluated gene and cell therapies. In parallel, growing attention has focused on both native and engineered roles of CRISPR-Cas in shaping HGT, plasmid ecology, and antimicrobial resistance (AMR), as AMR continues to expand globally. In this Review, we integrate advances spanning eukaryotic genome editing and prokaryotic antimicrobial applications. We summarize CRISPR-Cas classification and molecular mechanisms, highlighting spacer acquisition, guide RNA biogenesis, target recognition, and nucleic acid cleavage. We then examine how cellular DNA repair pathways influence editing outcomes and discuss strategies to enhance precision. We further review delivery strategies, such as conjugative plasmids, bacteriophages and phagemids, extracellular vesicles, and nanoparticles, together with evolutionary countermeasures encoded by mobile genetic elements, including anti-CRISPR proteins. Finally, we outline current limitations.