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The CRISPR-Cas systems, identified initially as adaptive immune mechanisms in bacteria and archaea against viral threats, have rapidly evolved into transformative tools in genetic engineering and biotechnology. These RNA-guided systems are broadly classified into Class 1, comprising multi-subunit complexes, and Class 2, characterized by compact single-effector protein, such as Cas9, Cas12, and Cas13. Their remarkable structural and functional diversity enables microorganisms to adapt to diverse ecological niches, offering a vast repertoire of genome-editing strategies. Beyond their natural role in maintaining genome integrity and defense, CRISPR-Cas systems have been extensively repurposed for precise genome modification, transcriptional regulation, epigenetic editing, and nucleic acid detection. Recent advances in computational mining of microbial genomes and metagenomes have uncovered a broad range of novel CRISPR effectors with unique properties, distinct protospacer adjacent motif (PAM) requirements, RNA-targeting capabilities, miniature architectures, and promiscuous cleavage activities that significantly expand the molecular biology toolkit. The development of CRISPR-based technologies such as base editing, prime editing, gene knock-in/out, and live-cell DNA/RNA imaging exemplifies the versatility of these systems. Despite the challenges associated with delivering complex Class 1 systems, both classes are now being actively harnessed across diverse microbial platforms. Concurrently, the CRISPR-Cas research, particularly for guide RNA (gRNA) design and activity prediction, has revolutionized target specificity and editing efficiency. This review presents a comprehensive overview of CRISPR-Cas system diversity, their genomic landscape in microorganisms, and their cutting-edge biotechnological applications. It also emphasizes the transformative potential of CRISPR in synthetic biology, therapeutics, diagnostics, environmental remediation, and agriculture, while also addressing the ethical and biosafety considerations surrounding its deployment. As CRISPR-Cas systems continue to evolve, they stand at the forefront of innovations that bridge natural microbial immunity with engineered precision tools for next-generation biotechnology. Full article

The rise of multidrug-resistant (MDR) Pseudomonas aeruginosa (P. aeruginosa) presents a significant challenge to clinical treatment and environmental risks. This review delves into the complex mechanisms underlying MDR development in P. aeruginosa, such as genetic mutations, horizontal gene transfer (HGT), and the interaction between virulence factors and resistance genes. It evaluates current detection methods, from traditional bacteriology to advanced molecular techniques, emphasizing the need for rapid and accurate diagnostics. This review also examines therapeutic strategies, including broad-spectrum antibiotics, novel drug candidates, combination therapies, and innovative approaches like RNA interference, CRISPR-Cas9 gene editing, and bridge RNA-guided gene editing. Importantly, this review highlights the distribution, migration, and environmental risks of MDR P. aeruginosa, underscoring its adaptability to diverse environments. It concludes by stressing the necessity for continued research and development in antimicrobial resistance, advocating for an integrated approach that combines genomics, clinical practice, and environmental considerations to devise innovative solutions and preserve antibiotic efficacy. Full article