CRISPR-Cas based genome editing for eradication of human viruses.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system possess a broad range of applications for genetic modification, diagnosis and treatment of infectious as well as non-infectious disease. The CRISPR-Cas system is found in bacteria and archaea that possess the Cas protein and guide RNA (gRNA). Cas9 and gRNA forms a complex to target and cleave the desired gene, providing defense against viral infections. Human immunodeficiency virus (HIV), hepatitis B virus (HBV), herpesviruses, human papillomavirus (HPV), and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) cause major life threatening diseases which cannot cure completely by drugs. This chapter describes the present strategy of CRISPR-Cas systems for altering the genomes of viruses, mostly human ones, in order to control infections.

Progress on CRISPR-Cas gene editing technology in sheep production.

Gene editing is a kind of genetic engineering technology that can modify the genome. In recent years, with the rapid development of molecular biotechnology, the clustered regularly interspaced short palindromic repeats associated protein system has been widely used as a powerful gene editing tool due to its high efficiency, accuracy and flexibility. The CRISPR-Cas system makes a significant contribution to different aspects of livestock production by introducing site-specific modifications such as insertions, deletions or single base replacements at specific genomic sites. In terms of sheep production applications, by establishing animal models that improve production economic traits and disease resistance, the function of key genes can be studied to accelerate the improvement of traits, thereby accelerating the improvement of traits. In this review, we summarize the mechanism and function of CRISPR-Cas system and its application in the production of reproductive traits, meat use traits, wool production traits, lactation traits and disease resistance traits of sheep and the establishment of sheep animal models.

Commercial Strip-Inspired One-Pot CRISPR-Based Chip for Multiplexed Detection of Respiratory Viruses.

The absence of sensitive, multiplexed, and point-of-care assays poses a critical obstacle in promptly responding to emerging human respiratory virus (HRV) pandemics. Herein, RECOGNIZER (re-building commercial pregnancy strips via large-size nanoflowers), an innovative one-pot CRISPR assay, is presented that employs commercially available strips to identify several types of HRVs. The superiority of the RECOGNIZER assay mainly relies on two aspects: (i) DNA nanoflowers possessing a high surface-to-volume ratio and well-defined surface allow for a considerable probe loading density and minimized non-specific interaction, achieving an impressive signal-to-noise proportion exceeding tenfold at 1 nM target. (ii) The design of the one-pot reaction, multi-channel chip, and custom-made app enables the rapid, sample-to-answer, and multiplexed analysis of four HRVs in 25 min. This assay demonstrates a sensitivity of 5.42 pM for synthetic SARS-CoV-2 RNA and 10 copies µL-1 for SARS-CoV-2 plasmids after pre-amplification. Finally, the proposed approach indicated 100% accuracy in 50 clinical swab samples, demonstrating the robust performance in distinguishing SARS-CoV-2 from other HRVs. The versatility and scalability of RECOGNIZER renders it a user-friendly platform for virus infection monitoring, offering significant potential for improving pandemic response efforts.

New Viruses Infecting Hyperthermophilic Bacterium Thermus thermophilus.

Highly diverse phages infecting thermophilic bacteria of the Thermus genus have been isolated over the years from hot springs around the world. Many of these phages are unique, rely on highly unusual developmental strategies, and encode novel enzymes. The variety of Thermus phages is clearly undersampled, as evidenced, for example, by a paucity of phage-matching spacers in Thermus CRISPR arrays. Using water samples collected from hot springs in the Kunashir Island from the Kuril archipelago and from the Tsaishi and Nokalakevi districts in the Republic of Georgia, we isolated several distinct phages infecting laboratory strains of Thermus thermophilus. Genomic sequence analysis of 11 phages revealed both close relatives of previously described Thermus phages isolated from geographically distant sites, as well as phages with very limited similarity to earlier isolates. Comparative analysis allowed us to predict several accessory phage genes whose products may be involved in host defense/interviral warfare, including a putative Type V CRISPR-cas system.

Role of CRISPR-Cas systems and anti-CRISPR proteins in bacterial antibiotic resistance.

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.

CRISPR-Based Biosensors for Medical Diagnosis: Readout from Detector-Dependence Detection Toward Naked Eye Detection.

The detection of biomarkers (such as DNA, RNA, and protein) plays a vital role in medical diagnosis. The CRISPR-based biosensors utilize the CRISPR/Cas system for biometric recognition of targets and use biosensor strategy to read out biological signals without the employment of professional operations. Consequently, the CRISPR-based biosensors demonstrate great potential for the detection of biomarkers with high sensitivity and specificity. However, the signal readout still relies on specialized detectors, limiting its application in on-site detection for medical diagnosis. In this review, we summarize the principles and advances of the CRISPR-based biosensors with a focus on medical diagnosis. Then, we review the advantages and progress of CRISPR-based naked eye biosensors, which can realize diagnosis without additional detectors for signal readout. Finally, we discuss the challenges and further prospects for the development of CRISPR-based biosensors.

Genomic analysis of two penicillin- and rifampin-resistant Corynebacterium rouxii strains isolated from cutaneous infections in dogs.

Although diphtheria is a vaccine-preventable disease, numerous cases are still reported around the world, as well as outbreaks in countries, including European ones. Species of the Corynebacterium diphtheriae complex are potentially toxigenic and, therefore, must be considered given the possible consequences, such as the circulation of clones and transmission of antimicrobial resistance and virulence genes. Recently, Corynebacterium rouxii was characterized and included among the valid species of the complex. Therefore, two cases of C. rouxii infection arising from infections in domestic animals are presented here. We provide molecular characterization, phylogenetic analyses, genome sequencing, and CRISPR-Cas analyses to contribute to a better understanding of the molecular bases, pathogenesis, and epidemiological monitoring of this species, which is still little studied. We confirmed its taxonomic position with genome sequencing and in silico analysis and identified the ST-918 for both strains. The clinical isolates were sensitive resistance to benzylpenicillin and rifampin. Antimicrobial resistance genes, including tetB, rpoB2, and rbpA genes, were predicted. The bla and ampC genes were not found. Several virulence factors were also detected, including adhesion, iron uptake systems, gene regulation (dtxR), and post-translational modification (MdbA). Finally, one prophage and the Type I-E CRISPR-Cas system were identified.

Trends in developing one-pot CRISPR diagnostics strategies.

The integration of nucleic acid amplification (NAA) with the CRISPR detection system has led to significant advancements and opportunities for development in molecular diagnostics. Nevertheless, the incompatibility between CRISPR cleavage and NAA has significantly impeded the commercialization of this technology. Currently, several one-pot detection strategies based on CRISPR systems have been devised to address concerns regarding aerosol contamination risk and operational complexity associated with step-by-step detection as well as the sensitivity limitation of conventional one-pot methods. In this review, we provide a comprehensive introduction and outlook of the various solutions of the one-pot CRISPR assay for practitioners who are committed to developing better CRISPR nucleic acid detection technologies to promote the progress of molecular diagnostics.

Efficient genome-editing tools to engineer the recalcitrant non-model industrial microorganism Zymomonas mobilis.

Current biotechnology relies on a few well-studied model organisms, such as Escherichia coli and Saccharomyces cerevisiae, for which abundant information and efficient toolkits are available for genetic manipulation, but which lack industrially favorable characteristics. Non-model industrial microorganisms usually do not have effective and/or efficient genome-engineering toolkits, which hampers the development of microbial cell factories to meet the fast-growing bioeconomy. In this study, using the non-model ethanologenic bacterium Zymomonas mobilis as an example, we developed a workflow to mine and temper the elements of restriction-modification (R-M), CRISPR/Cas, toxin-antitoxin (T-A) systems, and native plasmids, which are hidden within industrial microorganisms themselves, as efficient genome-editing toolkits, and established a genome-wide iterative and continuous editing (GW-ICE) system for continuous genome editing with high efficiency. This research not only provides tools and pipelines for engineering the non-model polyploid industrial microorganism Z. mobilis efficiently, but also sets a paradigm to overcome biotechnological limitations in other genetically recalcitrant non-model industrial microorganisms.

Infection by a multidrug-resistant Corynebacterium diphtheriae strain: prediction of virulence factors, CRISPR-Cas system analysis, and structural implications of mutations conferring rifampin resistance.

Cases of diphtheria, even in immunized individuals, are still reported in several parts of the world, including in Brazil. New outbreaks occur in Europe and other continents. In this context, studies on Corynebacterium diphtheriae infections are highly relevant, both for a better understanding of the pathogenesis of the disease and for controlling the circulation of clones and antimicrobial resistance genes. Here we present a case of cutaneous infection by multidrug-resistant Corynebacterium diphtheriae and provide its whole-genome sequencing. Genomic analysis revealed resistance genes, including tet(W), sul1, cmx, rpoB2, rbpA and mutation in rpoB. We performed phylogenetic analyzes and used the BRIG to compare the predicted resistance genes with those found in genomes from other significant isolates, including those associated with some outbreaks. Virulence factors such as spaD, srtBC, spaH, srtDE, surface-anchored pilus proteins (sapD), nonfimbrial adhesins (DIP0733, DIP1281, and DIP1621), embC and mptC (putatively involved in CdiLAM), sigA, dtxR and MdbA (putatively involved) in post-translational modification, were detected. We identified the CRISPR-Cas system in our isolate, which was classified as Type II-U based on the database and contains 15 spacers. This system functions as an adaptive immune mechanism. The strain was attributed to a new sequence type ST-928, and phylogenetic analysis confirmed that it was related to ST-634 of C. diphtheriae strains isolated in French Guiana and Brazil. In addition, since infections are not always reported, studies with the sequence data might be a way to complement and inform C. diphtheriae surveillance.

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

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 (K d = 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 preorder 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.

CRISPR-Cas3 and type I restriction-modification team up against blaKPC-IncF plasmid transfer in Klebsiella pneumoniae.

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.

CRISPR/Cas-Mediated Genome Engineering in Plants: Application and Prospectives.

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.

Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system continues to evolve, thereby enabling more precise detection and repair of mutagenesis. The development of CRISPR/Cas-based diagnosis holds promise for high-throughput, cost-effective, and portable nucleic acid screening and genetic disease diagnosis. In addition, advancements in transportation strategies such as adeno-associated virus (AAV), lentiviral vectors, nanoparticles, and virus-like vectors (VLPs) offer synergistic insights for gene therapeutics in vivo. Wilson's disease (WD), a copper metabolism disorder, is primarily caused by mutations in the ATPase copper transporting beta (ATP7B) gene. The condition is associated with the accumulation of copper in the body, leading to irreversible damage to various organs, including the liver, nervous system, kidneys, and eyes. However, the heterogeneous nature and individualized presentation of physical and neurological symptoms in WD patients pose significant challenges to accurate diagnosis. Furthermore, patients must consume copper-chelating medication throughout their lifetime. Herein, we provide a detailed description of WD and review the application of novel CRISPR-based strategies for its diagnosis and treatment, along with the challenges that need to be overcome.

Exploring non-viral methods for the delivery of CRISPR-Cas ribonucleoprotein to hematopoietic stem cells.

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.

[Application of the CRISPR/Cas system in gene editing and nucleic acid detection of parasitic diseases: a review].

CRISPR/Cas system, an adaptive immune system with clustered regularly interspaced short palindromic repeats, may interfere with exogenous nucleic acids and protect prokaryotes from external damages, is an effective gene editing and nucleic acid detection tools. The CRISPR/Cas system has been widely applied in virology and bacteriology; however, there is relatively less knowledge about the application of the CRISPR/Cas system in parasitic diseases. The review summarizes the mechanisms of action of the CRISPR/Cas system and provides a comprehensive overview of their application in gene editing and nucleic acid detection of parasitic diseases, so as to provide insights into future studies on parasitic diseases.

Occurrence and diversity pattern of CRISPR-Cas systems in Acetobacter genus provides insights on adaptive defense mechanisms against to invasive DNAs.

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.

Endogenous Type I-C CRISPR-Cas system of Streptococcus equi subsp. zooepidemicus promotes biofilm formation and pathogenicity.

Streptococcus equi subsp. zooepidemicus (SEZ) is a significant zoonotic pathogen that causes septicemia, meningitis, and mastitis in domestic animals. Recent reports have highlighted high-mortality outbreaks among swine in the United States. Traditionally recognized for its adaptive immune functions, the CRISPR-Cas system has also been implicated in gene regulation, bacterial pathophysiology, virulence, and evolution. The Type I-C CRISPR-Cas system, which is prevalent in SEZ isolates, appears to play a pivotal role in regulating the pathogenicity of SEZ. By constructing a Cas3 mutant strain (ΔCas3) and a CRISPR-deficient strain (ΔCRISPR), we demonstrated that this system significantly promotes biofilm formation and cell adhesion. However, the deficiency in the CRISPR-Cas system did not affect bacterial morphology or capsule production. In vitro studies showed that the CRISPR-Cas system enhances pro-inflammatory responses in RAW264.7 cells. The ΔCas3 and ΔCRISPR mutant strains exhibited reduced mortality rates in mice, accompanied by a decreased bacterial load in specific organs. RNA-seq analysis revealed distinct expression patterns in both mutant strains, with ΔCas3 displaying a broader range of differentially expressed genes, which accounted for over 70% of the differential genes observed in ΔCRISPR. These genes were predominantly linked to lipid metabolism, the ABC transport system, signal transduction, and quorum sensing. These findings enhance our understanding of the complex role of the CRISPR-Cas system in SEZ pathogenesis and provide valuable insights for developing innovative therapeutic strategies to combat infections.

Systematic metabolic engineering of Zymomonas mobilis for ß-farnesene production.

Zymomonas mobilis is an ethanologenic bacterium that can produce hopanoids using farnesyl pyrophosphate (FPP), which can be used as the precursor by ß-farnesene synthase for ß-farnesene production. To explore the possibility and bottlenecks of developing Z. mobilis for ß-farnesene production, five heterologous ß-farnesene synthases were selected and screened, and AaBFS from Artemisia annua had the highest ß-farnesene titer. Recombinant strains with AaBFS driven by the strong constitutive promoter Pgap (Pgap-AaBFS) doubled its ß-farnesene production to 25.73 ± 0.31 mg/L compared to the recombinant strain with AaBFS driven by Ptet (Ptet-AaBFS), which can be further improved by overexpressing the Pgap-AaBFS construct using the strategies of multiple plasmids (41.00 ± 0.40 mg/L) or genomic multi-locus integration (48.33 ± 3.40 mg/L). The effect of cofactor NADPH balancing on ß-farnesene production was also investigated, which can be improved only in zwf-overexpressing strains but not in ppnK-overexpressing strains, indicating that cofactor balancing is important and sophisticated. Furthermore, the ß-farnesene titer was improved to 73.30 ± 0.71 mg/L by overexpressing dxs, ispG, and ispH. Finally, the ß-farnesene production was increased to 159.70 ± 7.21 mg/L by fermentation optimization, including the C/N ratio, flask working volume, and medium/dodecane ratio, which was nearly 13-fold improved from the parental strain. This work thus not only generated a recombinant ß-farnesene production Z. mobilis strain but also unraveled the bottlenecks to engineer Z. mobilis for farnesene production, which will help guide the future rational design and construction of cell factories for terpenoid production in non-model industrial microorganisms.

Application of novel CRISPR tools in brain therapy.

In recent years, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based genome editing toolkit has been widely used to modify the genome sequence of organisms. As the CRISPR toolbox continues to grow and new CRISPR-associated (Cas) proteins are discovered, its applications have expanded beyond conventional genome editing. This now encompass epigenetic editing, gene expression control, and various other functions. Notably, these advancements are finding practical application in the treatment of brain diseases. Furthermore, the amalgamation of CRISPR and Chimeric Antigen Receptor T-cell (CAR-T) technologies has emerged as a potential approach for disease treatment. With this in mind, this review commences by offering a comprehensive overview of recent advancements in CRISPR gene editing tools. This encompasses an exploration of various Cas proteins, gene expression control, epigenetic editing, base editing and primer editing. Additionally, we present an in-depth examination of the manifold applications of these innovative CRISPR tools in the realms of brain therapeutics, such as neurodegenerative diseases, neurological syndromes and genetic disorders, epileptic disorders, and brain tumors, also explore the pathogenesis of these diseases. This includes their utilization in modeling, gene screening, therapeutic gene editing, as well as their emerging synergy with CAR-T technology. Finally, we discuss the remaining technical challenges that need to be addressed for effective utilization of CRISPR tools in disease treatment.