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CRISPR-Cas technologies have emerged as powerful and versatile tools in gene therapy. In addition to the widely used SpCas9 system, alternative platforms including modified amino acid sequences, size-optimized variants, and other Cas enzymes from diverse bacterial species have been developed to apply this technology in various genetic contexts. In addition, base editors and prime editors for precise gene editing, the Cas13 system targeting RNA, and CRISPRa/i systems have enabled diverse and adaptable approaches for genome and RNA editing, as well as for regulating gene expression. Typically, CRISPR-Cas components are transported to the target in the form of DNA, RNA, or ribonucleoprotein complexes using various delivery methods, such as electroporation, adeno-associated viruses, and lipid nanoparticles. To amplify therapeutic efficiency, continued developments in targeted delivery technologies are required, with increased safety and stability of therapeutic biomolecules. CRISPR-based therapeutics hold an inexhaustible potential for the treatment of many diseases, including rare congenital diseases, by making permanent corrections at the genomic DNA level. In this review, we present various CRISPR-based tools, their delivery systems, and clinical progress in the CRISPR-Cas technology, highlighting its innovative prospects for gene therapy.
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CRISPR-Cas9-based gene editing enables precise genetic modifications. However, its application to human cytomegalovirus (HCMV) remains challenging due to the large size of the viral genome and the essential roles of key regulatory genes. Here, we establish an optimized CRISPR-Cas9 system for precise labeling and functional analysis of HCMV immediate early (IE) genes. By integrating a multifunctional cassette encoding an auxin-inducible degron (AID), a self-cleaving peptide (P2A), and GFP into the viral genome via homology-directed repair (HDR), we achieved efficient knock-ins without reliance on bacterial artificial chromosome (BAC) cloning, a labor-intensive and time-consuming approach. We optimized delivery strategies, donor template designs, and component ratios to enhance HDR efficiency, significantly improving knock-in success rates. This system enables real-time fluorescent tracking and inducible protein degradation, allowing temporal control of essential viral proteins through auxin-mediated depletion. Our approach provides a powerful tool for dissecting the dynamic roles of viral proteins throughout the HCMV life cycle, facilitating a deeper understanding of viral pathogenesis and potential therapeutic targets.
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Spo0A, the master regulator of sporulation initiation in Bacillus subtilis, controls over 500 genes directly or indirectly in early sporulation stages. Although the effects of Spo0A disruption on sporulation have been extensively studied, a comprehensive understanding of the genomic response throughout growth phases remain elusive. Here, we examined the transcriptomic changes in Spo0A mutant strain, R211E, and wild-type across a time-course RNA-seq to identify impacted biological processes and pathways. The R211E strain, which exhibits sporulation deficiency, was constructed using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein (Cas)9 system, highlighting the critical role of proper Cas9 dosing in gene editing. Functional analysis of 3,010 differentially expressed genes (DEGs) showed significant alterations in sporulation, quorum sensing, metabolism, and biofilm formation. The R211E disrupted the Spo0A-AbrB regulatory pathway, reducing biofilm formation and enhancing flagellar gene expression. Up-regulated metabolic pathways, including glycolysis, histidine, and purine biosynthesis, increased cell numbers during vegetative growth. Further, the mutant displayed elevated vegetative autolysin expression, resulting in reduced cell viability in the stationary phase. We also introduce the novel potential of R211E in a recombinant protein expression system that facilitated protein release into the supernatant, providing valuable insight for future research in metabolic engineering and efficient production systems in B. subtilis.
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Phage specificity primarily relies on host cell-surface receptors. However, integrating cas genes and guide RNAs into phage genomes could enhance their target specificity and regulatory effects. In this study, we developed a CRISPR-Cas12f1 system-equipped bacteriophage λ model capable of detecting Escherichia coli target genes. We demonstrated that synthetic λ phages carrying Cas12f1-sgRNA can effectively prevent lysogen formation. Furthermore, we showcased that truncating the 3'-end of sgRNA enables precise identification of single-nucleotide variations in the host genome. Moreover, infecting E. coli strains carrying various stx2 gene subtypes encoding Shiga toxin with bacteriophages harboring Cas12f1 and truncated sgRNAs resulted in the targeted elimination of strains with matching subtype genes. These findings underscore the ability of phages equipped with the CRISPR-Cas12f1 system to precisely control microbial hosts by recognizing genomic sequences with high resolution.
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