The emergence and development of antibiotic resistance in bacteria is a serious threat to global public health. Antibiotic resistance genes (ARGs) are often located on mobile genetic elements (MGEs). They can be transferred among bacteria by horizontal gene transfer (HGT), leading to the spread of drug-resistant strains and antibiotic treatment failure. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated genes) is one of the many strategies bacteria have developed under long-term selection pressure to restrict the HGT. CRISPR-Cas systems exist in about half of bacterial genomes and play a significant role in limiting the spread of antibiotic resistance. On the other hand, bacteriophages and other MGEs encode a wide range of anti-CRISPR proteins (Acrs) to counteract the immunity of the CRISPR-Cas system. The Acrs could decrease the CRISPR-Cas system\'s activity against phages and facilitate the acquisition of ARGs and virulence traits for bacteria. This review aimed to assess the relationship between the CRISPR-Cas systems and Acrs with bacterial antibiotic resistance. We also highlighted the CRISPR technology and Acrs to control and prevent antibacterial resistance. The CRISPR-Cas system can target nucleic acid sequences with high accuracy and reliability; therefore, it has become a novel gene editing and gene therapy tool to prevent the spread of antibiotic resistance. CRISPR-based approaches may pave the way for developing smart antibiotics, which could eliminate multidrug-resistant (MDR) bacteria and distinguish between pathogenic and beneficial microorganisms. Additionally, the engineered anti-CRISPR gene-containing phages in combination with antibiotics could be used as a cutting-edge treatment approach to reduce antibiotic resistance.

Gene manipulation of hematopoietic stem cells (HSCs) using the CRISPR/Cas system as a potent genome editing tool holds immense promise for addressing hematologic disorders. An essential hurdle in advancing this treatment lies in effectively delivering CRISPR/Cas to HSCs. While various delivery formats exist, Ribonucleoprotein complex (RNP) emerges as a particularly efficient option. RNP complexes offer enhanced gene editing capabilities, devoid of viral vectors, with rapid activity and minimized off-target effects. Nevertheless, novel delivery methods such as microfluidic-based techniques, filtroporation, nanoparticles, and cell-penetrating peptides are continually evolving. This study aims to provide a comprehensive review of these methods and the recent research on delivery approaches of RNP complexes to HSCs.

Genetic engineering has become an essential element in developing climate-resilient crops and environmentally sustainable solutions to respond to the increasing need for global food security. Genome editing using CRISPR/Cas [Clustered regulatory interspaced short palindromic repeat (CRISPR)-associated protein (Cas)] technology is being applied to a variety of organisms, including plants. This technique has become popular because of its high specificity, effectiveness, and low production cost. Therefore, this technology has the potential to revolutionize agriculture and contribute to global food security. Over the past few years, increasing efforts have been seen in its application in developing higher-yielding, nutrition-rich, disease-resistant, and stress-tolerant \"crops\", fruits, and vegetables. Cas proteins such as Cas9, Cas12, Cas13, and Cas14, among others, have distinct architectures and have been used to create new genetic tools that improve features that are important for agriculture. The versatility of Cas has accelerated genomic analysis and facilitated the use of CRISPR/Cas to manipulate and alter nucleic acid sequences in cells of different organisms. This review provides the evolution of CRISPR technology exploring its mechanisms and contrasting it with traditional breeding and transgenic approaches to improve different aspects of stress tolerance. We have also discussed the CRISPR/Cas system and explored three Cas proteins that are currently known to exist: Cas12, Cas13, and Cas14 and their potential to generate foreign-DNA-free or non-transgenic crops that could be easily regulated for commercialization in most countries.

The Acetobacter genus is primarily known for its significance in acetic acid production and its application in various industrial processes. This study aimed to shed light on the prevalence, diversity, and functional implications of CRISPR-Cas systems in the Acetobacter genus using a genome mining approach. The investigation analyzed the CRISPR-Cas architectures and components of 34 Acetobacter species, as well as the evolutionary strategies employed by these bacteria in response to phage invasion and foreign DNA. Furthermore, phylogenetic analysis based on CAS1 protein sequences was performed to gain insights into the evolutionary relationships among Acetobacter strains, with an emphasis on the potential of this protein for genotyping purposes. The results showed that 15 species had orphan, while20 species had complete CRISPR-Cas systems, resulting in an occurrence rate of 38% for complete systems in Acetobacter strains. The predicted complete CRISPR-Cas systems were categorized into I-C, I-F, I-E, and II-C subtypes, with subtype I-E being the most prevalent in Acetobacter. Additionally, spacer homology analysis revealed against such the dynamic interaction between Acetobacter strains and foreign invasive DNAs, emphasizing the pivotal role of CRISPR-Cas systems in defending against such invasions. Furthermore, the investigation of the secondary structures of CRISPR arrays revealed the conserved patterns within subtypes despite variations in repeat sequences. The exploration of protospacer adjacent motifs (PAMs) identified distinct recognition motifs in the flanking regions of protospacers. In conclusion, this research not only contributes to the growing body of knowledge on CRISPR-Cas systems but also establishes a foundation for future studies on the adaptive defense mechanisms of Acetobacter. The findings provide valuable insights into the intricate interplay between bacteria and phages, with implications for industrial applications and potential biotechnological advancements.

CRISPR-Cas12a binds and processes a single pre-crRNA during maturation, providing a simple tool for genome editing applications. Here, we constructed a kinetic and thermodynamic framework for pre-crRNA processing by Cas12a in vitro, and we measured the contributions of distinct regions of the pre-crRNA to this reaction. We find that the pre-crRNA binds rapidly and extraordinarily tightly to Cas12a (Kd = 0.6 pM), such that pre-crRNA binding is fully rate limiting for processing and therefore determines the specificity of Cas12a for different pre-crRNAs. The guide sequence contributes 10-fold to the binding affinity of the pre-crRNA, while deletion of an upstream sequence has no significant effect. After processing, the mature crRNA remains very tightly bound to Cas12a with a comparable affinity. Strikingly, the affinity contribution of the guide region increases to 600-fold after processing, suggesting that additional contacts are formed and may pre-order the crRNA for efficient DNA target recognition. Using a direct competition assay, we find that pre-crRNA binding specificity is robust to changes in the guide sequence, addition of a 3\' extension, and secondary structure within the guide region. However, stable secondary structure in the guide region can strongly inhibit DNA targeting, indicating that care should be taken in crRNA design. Together our results provide a quantitative framework for pre-crRNA binding and processing by Cas12a and suggest strategies for optimizing crRNA design in genome editing applications.

OBJECTIVE: We explored whether the Clustered regularly interspaced short palindromic repeat (CRISPR)-Cas and restriction-modification (R-M) systems are compatible and act together to resist plasmid attacks.
METHODS: 932 global whole-genome sequences from GenBank, and 459 K. pneumoniae isolates from six provinces of China, were collected to investigate the co-distribution of CRISPR-Cas, R-M systems, and blaKPC plasmid. Conjugation and transformation assays were applied to explore the anti-plasmid function of CRISPR and R-M systems.
RESULTS: We found a significant inverse correlation between the presence of CRISPR and R-M systems and blaKPC plasmids in K. pneumoniae, especially when both systems cohabited in one host. The multiple matched recognition sequences of both systems in blaKPC-IncF plasmids (97%) revealed that they were good targets for both systems. Furthermore, the results of conjugation assay demonstrated that CRISPR-Cas and R-M systems in K. pneumoniae could effectively hinder blaKPC plasmid invasion. Notably, CRISPR-Cas and R-M worked together to confer a 4-log reduction in the acquisition of blaKPC plasmid in conjugative events, exhibiting robust synergistic anti-plasmid immunity.
CONCLUSIONS: Our results indicate the synergistic role of CRISPR and R-M in regulating horizontal gene transfer in K. pneumoniae and rationalize the development of antimicrobial strategies that capitalize on the immunocompromised status of KPC-KP.

UNASSIGNED: CRISPR-Cas is a prokaryotic adaptive immune system that protects bacteria and archaea against mobile genetic elements (MGEs) such as bacteriophages plasmids, and transposons. In this study, we aimed to assess the prevalence of the CRISPR-Cas systems and their association with antibiotic resistance in one of the most challenging bacterial pathogens, Klebsiella pneumoniae.
METHODS: A total of 105 K. pneumoniae isolates were collected from various clinical infections. Extended-spectrum β-lactamases (ESBLs) phenotypically were detected and the presence of ESBL, aminoglycoside-modifying enzymes (AME), and CRISPR-Cas system subtype genes were identified using PCR. Moreover, the diversity of the isolates was determined by enterobacterial repetitive intergenic consensus (ERIC)-PCR.
RESULTS: Phenotypically, 41.9% (44/105) of the isolates were found to be ESBL producers. A significant inverse correlation existed between the subtype I-E CRISPR-Cas system\'s presence and ESBL production in K. pneumoniae isolates. Additionally, the frequency of the ESBL genes blaCTX-M1 (3%), blaCTX-M9 (12.1%), blaSHV (51.5%), and blaTEM (33.3%), as well as some AME genes such as aac(3)-Iva (21.2%) and ant(2\'\')-Ia (3%) was significantly lower in the isolates with the subtype I-E CRISPR-Cas system in comparison to CRISPR-negative isolates. There was a significant inverse correlation between the presence of ESBL and some AME genes with subtype I-E CRISPR-Cas system.
CONCLUSIONS: The presence of the subtype I-E CRISPR-Cas system was correlated with the antibiotic-resistant gene (ARGs). The isolates with subtype I-E CRISPR-Cas system had a lower frequency of ESBL genes and some AME genes than CRISPR-negative isolates.

Target cancer therapy has been developed for clinical cancer treatment based on the discovery of CRISPR (clustered regularly interspaced short palindromic repeat) -Cas system. This forefront and cutting-edge scientific technique improves the cancer research into molecular level and is currently widely utilized in genetic investigation and clinical precision cancer therapy. In this review, we summarized the genetic modification by CRISPR/Cas and CRISPR screening system, discussed key components for successful CRISPR screening, including Cas enzymes, guide RNA (gRNA) libraries, target cells or organs. Furthermore, we focused on the application for CAR-T cell therapy, drug target, drug screening, or drug selection in both ex vivo and in vivo with CRISPR screening system. In addition, we elucidated the advantages and potential obstacles of CRISPR system in precision clinical medicine and described the prospects for future genetic therapy.In summary, we provide a comprehensive and practical perspective on the development of CRISPR/Cas and CRISPR screening system for the treatment of cancer defects, aiming to further improve the precision and accuracy for clinical treatment and individualized gene therapy.

Antimicrobial resistance poses a significant global health threat, necessitating innovative approaches for combatting it. This review explores various mechanisms of antimicrobial resistance observed in various strains of bacteria. We examine various strategies, including antimicrobial peptides (AMPs), novel antimicrobial materials, drug delivery systems, vaccines, antibody therapies, and non-traditional antibiotic treatments. Through a comprehensive literature review, the efficacy and challenges of these strategies are evaluated. Findings reveal the potential of AMPs in combating resistance due to their unique mechanisms and lower propensity for resistance development. Additionally, novel drug delivery systems, such as nanoparticles, show promise in enhancing antibiotic efficacy and overcoming resistance mechanisms. Vaccines and antibody therapies offer preventive measures, although challenges exist in their development. Non-traditional antibiotic treatments, including CRISPR-Cas systems, present alternative approaches to combat resistance. Overall, this review underscores the importance of multifaceted strategies and coordinated global efforts to address antimicrobial resistance effectively.

As a response to viral infections, bacteria have evolved the CRISPR-Cas system as an adaptive immune mechanism, enabling them to target and eliminate viral genetic material introduced during infection. However, viruses have also evolved mechanisms to counteract this bacterial defense, including anti-CRISPR proteins, which can inactivate the CRISPR-Cas adaptive immune system, thus aiding the viruses in their survival and replication within bacterial hosts. In this study, we establish the high-resolution crystal structure of the Type IE anti-CRISPR protein, AcrIE3. Our structural examination showed that AcrIE3 adopts a helical bundle fold comprising four α-helices, with a notably extended loop at the N-terminus. Additionally, surface analysis of AcrIE3 revealed the presence of three acidic regions, which potentially play a crucial role in the inhibitory function of this protein. The structural information we have elucidated for AcrIE3 will provide crucial insights into fully understanding its inhibitory mechanism. Furthermore, this information is anticipated to be important for the application of the AcrIE family in genetic editing, paving the way for advancements in gene editing technologies.