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1
Roslan, Rozieffa, Peer Mohamed Abdul, and Jamaliah Md Jahim. "Endogenous CRISPR/Cas Systems Prediction: A Glimpse towards Harnessing CRISPR/ Cas Machineries for Genetic Engineering." Jurnal Kejuruteraan si1, no. 7 (November 30, 2018): 1–9. http://dx.doi.org/10.17576/jkukm-2018-si1(7)-01.
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Genetic engineering field has become an imperative approach for enhancement of various bioproducts yield and productivity; and found extended applications in gene therapy, nanotechnology, as well as industrial microbiology. Modern genetic engineering tool CRISPR/Cas system, specifically the Type II system from Streptococcus pyogenes, is gaining traction in recent years and being utilized to engineer novel strains to overproduce primary fermentation product of interest. Employing this technology for non-model microorganism such as Clostridium spp is still restricted due to several limitations such as inadequate genome information, resistance against transformation, low plasmid replication, and the ability for gene expression. The prediction of CRISPR/Cas systems in microbial genomes is fundamentally the initial step towards exploitation of this technology to engineer Clostridium spp. In this study, we demonstrate a simple yet effective method to predict component of endogenous CRISPR/Cas systems, using Clostridium spp genomes as a proof-of-concept. We identified the “real” CRISPR array together with the cas gene operon consist of Type I B signature proteins in Clostridium pasteurianum which is in agreement with the previous report, implying that this strategy generates reliable CRISPR/Cas systems prediction. Thus, this provides a glimpse on how bioinformatics and biocomputational tools can be utilized to overcome barriers in genetic engineering.
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Shmakov, Sergey A., Kira S. Makarova, Yuri I. Wolf, Konstantin V. Severinov, and Eugene V. Koonin. "Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): E5307—E5316. http://dx.doi.org/10.1073/pnas.1803440115.
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The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of direct repeat arrays separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate all stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all CRISPR-Cas systems of a given (sub)type and are essential for the defense function, numerous genes occur in CRISPR-cas loci only sporadically. Some of these have been shown to perform various ancillary roles in CRISPR response, but the functional relevance of most remains unknown. We developed a computational strategy for systematically detecting genes that are likely to be functionally linked to CRISPR-Cas. The approach is based on a “CRISPRicity” metric that measures the strength of CRISPR association for all protein-coding genes from sequenced bacterial and archaeal genomes. Uncharacterized genes with CRISPRicity values comparable to those of cas genes are considered candidate CRISPR-linked genes. We describe additional criteria to predict functionally relevance for genes in the candidate set and identify 79 genes as strong candidates for functional association with CRISPR-Cas systems. A substantial majority of these CRISPR-linked genes reside in type III CRISPR-cas loci, which implies exceptional functional versatility of type III systems. Numerous candidate CRISPR-linked genes encode integral membrane proteins suggestive of tight membrane association of CRISPR-Cas systems, whereas many others encode proteins implicated in various signal transduction pathways. These predictions provide ample material for improving annotation of CRISPR-cas loci and experimental characterization of previously unsuspected aspects of CRISPR-Cas system functionality.
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Zhuang, Xiwei, Xueqiong Yang, Bo Cao, Haiming Sun, Xiaoyan Lv, Chijia Zeng, Fugang Li, et al. "Review—CRISPR/Cas Systems: Endless Possibilities for Electrochemical Nucleic Acid Sensors." Journal of The Electrochemical Society 169, no. 3 (March 1, 2022): 037522. http://dx.doi.org/10.1149/1945-7111/ac5cec.
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The CRISPR/Cas system has gained enormous attention for its excellent gene-editing capabilities. In recent years, the reported trans-cleavage activity of some Cas proteins, including Cas12, Cas13 and Cas14, has given the CRISPR/Cas system an increasingly powerful molecular diagnostic ability. When the CRISPR/Cas system is introduced into the field of electrochemical (EC) biosensor, it confers the high specificity to distinguish single base mismatches of nucleic acid, excellent sensitivity with the limit of detection as low as attomole range, and well meets the point-of-care testing (POCT) requirements of nucleic acid testing (NAT). In this review, we have briefly introduced the history and inherent advantages of the CRISPR/Cas system. The EC sensing platforms based on CRISPR/Cas systems have been compared with the classical fluorescence and colorimetric platforms. And the isothermal amplification strategies suitable for CRISPR/Cas system have been summarized. After that, we have highlighted the application of EC biosensor based on CRISPR/Cas system (EC-CRISPR) in the detection and identification of cancers, bacteria and viruses. Finally, the future prospects of EC-CRISPR have been proposed.
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Li, Ming, Luyao Gong, Feiyue Cheng, Haiying Yu, Dahe Zhao, Rui Wang, Tian Wang, et al. "Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems." Science 372, no. 6541 (April 29, 2021): eabe5601. http://dx.doi.org/10.1126/science.abe5601.
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CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA–resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.
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Shehreen, Saadlee, Te-yuan Chyou, Peter C. Fineran, and Chris M. Brown. "Genome-wide correlation analysis suggests different roles of CRISPR-Cas systems in the acquisition of antibiotic resistance genes in diverse species." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (March 25, 2019): 20180384. http://dx.doi.org/10.1098/rstb.2018.0384.
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CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa , the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Yang, Shanshan, Jian Huang, and Bifang He. "CASPredict: a web service for identifying Cas proteins." PeerJ 9 (July 30, 2021): e11887. http://dx.doi.org/10.7717/peerj.11887.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins constitute the CRISPR-Cas systems, which play a key role in prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas systems have also been designed to facilitate target gene editing in eukaryotic genomes. As one of the important components of the CRISPR-Cas system, Cas protein plays an irreplaceable role. The effector module composed of Cas proteins is used to distinguish the type of CRISPR-Cas systems. Effective prediction and identification of Cas proteins can help biologists further infer the type of CRISPR-Cas systems. Moreover, the class 2 CRISPR-Cas systems are gradually applied in the field of genome editing. The discovery of Cas protein will help provide more candidates for genome editing. In this paper, we described a web service named CASPredict (http://i.uestc.edu.cn/caspredict/cgi-bin/CASPredict.pl) for identifying Cas proteins. CASPredict first predicts Cas proteins based on support vector machine (SVM) by using the optimal dipeptide composition and then annotates the function of Cas proteins based on the hmmscan search algorithm. The ten-fold cross-validation results showed that the 84.84% of Cas proteins were correctly classified. CASPredict will be a useful tool for the identification of Cas proteins, or at least can play a complementary role to the existing methods in this area.
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Burmistrz, Michał, and Krzysztof Pyrc. "CRISPR-Cas Systems in Prokaryotes." Polish Journal of Microbiology 64, no. 3 (September 18, 2015): 193–202. http://dx.doi.org/10.5604/01.3001.0009.2114.
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Prokaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools.
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Li, Junwei, Yuexia Wang, Bin Wang, Juan Lou, Peng Ni, Yuefei Jin, Shuaiyin Chen, Guangcai Duan, and Rongguang Zhang. "Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases." Diagnostics 12, no. 10 (October 11, 2022): 2455. http://dx.doi.org/10.3390/diagnostics12102455.
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The CRISPR/Cas system is a protective adaptive immune system against attacks from foreign mobile genetic elements. Since the discovery of the excellent target-specific sequence recognition ability of the CRISPR/Cas system, the CRISPR/Cas system has shown excellent performance in the development of pathogen nucleic-acid-detection technology. In combination with various biosensing technologies, researchers have made many rapid, convenient, and feasible innovations in pathogen nucleic-acid-detection technology. With an in-depth understanding and development of the CRISPR/Cas system, it is no longer limited to CRISPR/Cas9, CRISPR/Cas12, and other systems that had been widely used in the past; other CRISPR/Cas families are designed for nucleic acid detection. We summarized the application of CRISPR/Cas-related technology in infectious-disease detection and its development in SARS-CoV-2 detection.
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Yang, Hui, and Dinshaw J. Patel. "New CRISPR-Cas systems discovered." Cell Research 27, no. 3 (February 21, 2017): 313–14. http://dx.doi.org/10.1038/cr.2017.21.
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Sternberg, Samuel H., Hagen Richter, Emmanuelle Charpentier, and Udi Qimron. "Adaptation in CRISPR-Cas Systems." Molecular Cell 61, no. 6 (March 2016): 797–808. http://dx.doi.org/10.1016/j.molcel.2016.01.030.
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Gophna, Uri, and Avital Brodt. "CRISPR/Cas systems in archaea." Mobile Genetic Elements 2, no. 1 (January 2012): 63–64. http://dx.doi.org/10.4161/mge.19907.
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Zhang, Jingfang, Li Chen, Ju Zhang, and Yu Wang. "Drug Inducible CRISPR/Cas Systems." Computational and Structural Biotechnology Journal 17 (2019): 1171–77. http://dx.doi.org/10.1016/j.csbj.2019.07.015.
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Wada, Naoki, Keishi Osakabe, and Yuriko Osakabe. "Expanding the plant genome editing toolbox with recently developed CRISPR–Cas systems." Plant Physiology 188, no. 4 (January 31, 2022): 1825–37. http://dx.doi.org/10.1093/plphys/kiac027.
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Abstract Since its first appearance, CRISPR–Cas9 has been developed extensively as a programmable genome-editing tool, opening a new era in plant genome engineering. However, CRISPR–Cas9 still has some drawbacks, such as limitations of the protospacer-adjacent motif (PAM) sequence, target specificity, and the large size of the cas9 gene. To combat invading bacterial phages and plasmid DNAs, bacteria and archaea have diverse and unexplored CRISPR–Cas systems, which have the potential to be developed as a useful genome editing tools. Recently, discovery and characterization of additional CRISPR–Cas systems have been reported. Among them, several CRISPR–Cas systems have been applied successfully to plant and human genome editing. For example, several groups have achieved genome editing using CRISPR–Cas type I-D and type I-E systems, which had never been applied for genome editing previously. In addition to higher specificity and recognition of different PAM sequences, recently developed CRISPR–Cas systems often provide unique characteristics that differ from well-known Cas proteins such as Cas9 and Cas12a. For example, type I CRISPR–Cas10 induces small indels and bi-directional long-range deletions ranging up to 7.2 kb in tomatoes (Solanum lycopersicum L.). Type IV CRISPR–Cas13 targets RNA, not double-strand DNA, enabling highly specific knockdown of target genes. In this article, we review the development of CRISPR–Cas systems, focusing especially on their application to plant genome engineering. Recent CRISPR–Cas tools are helping expand our plant genome engineering toolbox.
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Hryhorowicz, Magdalena, Daniel Lipiński, and Joanna Zeyland. "Evolution of CRISPR/Cas Systems for Precise Genome Editing." International Journal of Molecular Sciences 24, no. 18 (September 18, 2023): 14233. http://dx.doi.org/10.3390/ijms241814233.
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The bacteria-derived CRISPR/Cas (an acronym for regularly interspaced short palindromic repeats/CRISPR-associated protein) system is currently the most widely used, versatile, and convenient tool for genome engineering. CRISPR/Cas-based technologies have been applied to disease modeling, gene therapies, transcriptional modulation, and diagnostics. Nevertheless, some challenges remain, such as the risk of immunological reactions or off-target effects. To overcome these problems, many new methods and CRISPR/Cas-based tools have been developed. In this review, we describe the current classification of CRISPR systems and new precise genome-editing technologies, summarize the latest applications of this technique in several fields of research, and, finally, discuss CRISPR/Cas system limitations, ethical issues, and challenges.
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Chabas, Hélène, Viktor Müller, Sebastian Bonhoeffer, and Roland R. Regoes. "Epidemiological and evolutionary consequences of different types of CRISPR-Cas systems." PLOS Computational Biology 18, no. 7 (July 26, 2022): e1010329. http://dx.doi.org/10.1371/journal.pcbi.1010329.
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Bacteria have adaptive immunity against viruses (phages) in the form of CRISPR-Cas immune systems. Currently, 6 types of CRISPR-Cas systems are known and the molecular study of three of these has revealed important molecular differences. It is unknown if and how these molecular differences change the outcome of phage infection and the evolutionary pressure the CRISPR-Cas systems faces. To determine the importance of these molecular differences, we model a phage outbreak entering a population defending exclusively with a type I/II or a type III CRISPR-Cas system. We show that for type III CRISPR-Cas systems, rapid phage extinction is driven by the probability to acquire at least one resistance spacer. However, for type I/II CRISPR-Cas systems, rapid phage extinction is characterized by an a threshold-like behaviour: any acquisition probability below this threshold leads to phage survival whereas any acquisition probability above it, results in phage extinction. We also show that in the absence of autoimmunity, high acquisition rates evolve. However, when CRISPR-Cas systems are prone to autoimmunity, intermediate levels of acquisition are optimal during a phage outbreak. As we predict an optimal probability of spacer acquisition 2 factors of magnitude above the one that has been measured, we discuss the origin of such a discrepancy. Finally, we show that in a biologically relevant parameter range, a type III CRISPR-Cas system can outcompete a type I/II CRISPR-Cas system with a slightly higher probability of acquisition.
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Davidson, Alan R., Wang-Ting Lu, Sabrina Y. Stanley, Jingrui Wang, Marios Mejdani, Chantel N. Trost, Brian T. Hicks, Jooyoung Lee, and Erik J. Sontheimer. "Anti-CRISPRs: Protein Inhibitors of CRISPR-Cas Systems." Annual Review of Biochemistry 89, no. 1 (June 20, 2020): 309–32. http://dx.doi.org/10.1146/annurev-biochem-011420-111224.
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Clustered regularly interspaced short palindromic repeats (CRISPR) together with their accompanying cas (CRISPR-associated) genes are found frequently in bacteria and archaea, serving to defend against invading foreign DNA, such as viral genomes. CRISPR-Cas systems provide a uniquely powerful defense because they can adapt to newly encountered genomes. The adaptive ability of these systems has been exploited, leading to their development as highly effective tools for genome editing. The widespread use of CRISPR-Cas systems has driven a need for methods to control their activity. This review focuses on anti-CRISPRs (Acrs), proteins produced by viruses and other mobile genetic elements that can potently inhibit CRISPR-Cas systems. Discovered in 2013, there are now 54 distinct families of these proteins described, and the functional mechanisms of more than a dozen have been characterized in molecular detail. The investigation of Acrs is leading to a variety of practical applications and is providing exciting new insight into the biology of CRISPR-Cas systems.
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Koonin, Eugene V., and Kira S. Makarova. "Origins and evolution of CRISPR-Cas systems." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (March 25, 2019): 20180087. http://dx.doi.org/10.1098/rstb.2018.0087.
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CRISPR-Cas, the bacterial and archaeal adaptive immunity systems, encompass a complex machinery that integrates fragments of foreign nucleic acids, mostly from mobile genetic elements (MGE), into CRISPR arrays embedded in microbial genomes. Transcripts of the inserted segments (spacers) are employed by CRISPR-Cas systems as guide (g)RNAs for recognition and inactivation of the cognate targets. The CRISPR-Cas systems consist of distinct adaptation and effector modules whose evolutionary trajectories appear to be at least partially independent. Comparative genome analysis reveals the origin of the adaptation module from casposons, a distinct type of transposons, which employ a homologue of Cas1 protein, the integrase responsible for the spacer incorporation into CRISPR arrays, as the transposase. The origin of the effector module(s) is far less clear. The CRISPR-Cas systems are partitioned into two classes, class 1 with multisubunit effectors, and class 2 in which the effector consists of a single, large protein. The class 2 effectors originate from nucleases encoded by different MGE, whereas the origin of the class 1 effector complexes remains murky. However, the recent discovery of a signalling pathway built into the type III systems of class 1 might offer a clue, suggesting that type III effector modules could have evolved from a signal transduction system involved in stress-induced programmed cell death. The subsequent evolution of the class 1 effector complexes through serial gene duplication and displacement, primarily of genes for proteins containing RNA recognition motif domains, can be hypothetically reconstructed. In addition to the multiple contributions of MGE to the evolution of CRISPR-Cas, the reverse flow of information is notable, namely, recruitment of minimalist variants of CRISPR-Cas systems by MGE for functions that remain to be elucidated. Here, we attempt a synthesis of the diverse threads that shed light on CRISPR-Cas origins and evolution.This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Makarova, Kira S., Yuri I. Wolf, and Eugene V. Koonin. "The basic building blocks and evolution of CRISPR–Cas systems." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1392–400. http://dx.doi.org/10.1042/bst20130038.
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CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) is an adaptive immunity system in bacteria and archaea that functions via a distinct self/non-self recognition mechanism that involves unique spacers homologous with viral or plasmid DNA and integrated into the CRISPR loci. Most of the Cas proteins evolve under relaxed purifying selection and some underwent dramatic structural rearrangements during evolution. In many cases, CRISPR–Cas system components are replaced either by homologous or by analogous proteins or domains in some bacterial and archaeal lineages. However, recent advances in comparative sequence analysis, structural studies and experimental data suggest that, despite this remarkable evolutionary plasticity, all CRISPR–Cas systems employ the same architectural and functional principles, and given the conservation of the principal building blocks, share a common ancestry. We review recent advances in the understanding of the evolution and organization of CRISPR–Cas systems. Among other developments, we describe for the first time a group of archaeal cas1 gene homologues that are not associated with CRISPR–Cas loci and are predicted to be involved in functions other than adaptive immunity.
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Peretolchina, N. P., A. Y. Borisenko, Yu P. Dzhioev, and V. I. Zlobin. "COMPARATIVE ANALYSIS OF CRISPR-CAS SYSTEM STRUCTURES OF YERSINIA PSEUDOTUBERCULOSIS IP32953 AND IP31758." Acta Biomedica Scientifica 3, no. 5 (October 29, 2018): 54–59. http://dx.doi.org/10.29413/abs.2018-3.5.8.
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Background. Pseudotuberculosis is still relevant problem in medical science and public health of Russia and other countries. Typing of Y. рseudotuberculosis strains by their CRISPR systems is a perspective tool for monitoring of Yersinia populations as was shown in Y. pestis.Aims. Here we describe and compare CRISPR-Cas systems of Yersinia pseudotuberculosis strains IP32953 and IP31758 causing classic pseudotuberculosis and Far-East scarlet-like fever (FESLF) respectively.Materials and methods. Complete genomes of Y. pseudotuberculosis IP329353 and IP31758 (NC_006155 and NC_009708 respectively) were obtained from NCBI Nucleotide Database. Search; identification; and analysis of CRISPR systems were carried out by online-tools CRISPROne; CRISPRDetect; and CRISPRTarget.Results and discussion. Analyzed strains have CRISPR-Cas systems that include one set of cas-genes and arrays situated at the long distances from each other. We defined three CRISPR arrays in Y. pseudotuberculosis IP32953 by the combination of program methods. CRISPR-Cas system of this strain consist of array YP1 located near cas-genes; arrays YP2 and YP3. CRISPR-Cas system of Y. pseudotuberculosis IP31758 includes two arrays – YP1 and YP3. CRISPR systems do not share similar spacers. CRISPR systems of the analyzed strains differ in CRISPR loci and cas-protein structures that can be used as specific marks of analyzed strains.Conclusions. We suggest that acquisition of certain spacers may play a role in evolution and divergence of Y. pseudotuberculosis strains.
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Xu, Zeling, Shuzhen Chen, Weiyan Wu, Yongqi Wen, and Huiluo Cao. "Type I CRISPR-Cas-mediated microbial gene editing and regulation." AIMS Microbiology 9, no. 4 (2023): 780–800. http://dx.doi.org/10.3934/microbiol.2023040.
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<abstract> <p>There are six major types of CRISPR-Cas systems that provide adaptive immunity in bacteria and archaea against invasive genetic elements. The discovery of CRISPR-Cas systems has revolutionized the field of genetics in many organisms. In the past few years, exploitations of the most abundant class 1 type I CRISPR-Cas systems have revealed their great potential and distinct advantages to achieve gene editing and regulation in diverse microorganisms in spite of their complicated structures. The widespread and diversified type I CRISPR-Cas systems are becoming increasingly attractive for the development of new biotechnological tools, especially in genetically recalcitrant microbial strains. In this review article, we comprehensively summarize recent advancements in microbial gene editing and regulation by utilizing type I CRISPR-Cas systems. Importantly, to expand the microbial host range of type I CRISPR-Cas-based applications, these structurally complicated systems have been improved as transferable gene-editing tools with efficient delivery methods for stable expression of CRISPR-Cas elements, as well as convenient gene-regulation tools with the prevention of DNA cleavage by obviating deletion or mutation of the Cas3 nuclease. We envision that type I CRISPR-Cas systems will largely expand the biotechnological toolbox for microbes with medical, environmental and industrial importance.</p> </abstract>
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Parra-Sánchez, Ángel, Laura Antequera-Zambrano, Gema Martínez-Navarrete, Vanessa Zorrilla-Muñoz, José Luis Paz, Ysaias J. Alvarado, Lenin González-Paz, and Eduardo Fernández. "Comparative Analysis of CRISPR-Cas Systems in Pseudomonas Genomes." Genes 14, no. 7 (June 25, 2023): 1337. http://dx.doi.org/10.3390/genes14071337.
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Pseudomonas is a bacterial genus with some saprophytic species from land and others associated with opportunistic infections in humans and animals. Factors such as pathogenicity or metabolic aspects have been related to CRISPR-Cas, and in silico studies into it have focused more on the clinical and non-environmental setting. This work aimed to perform an in silico analysis of the CRISPR-Cas systems present in Pseudomonas genomes. It analyzed 275 complete genomic sequences of Pseudomonas taken from the NCBI database. CRISPR loci were obtained from CRISPRdb. The genes associated with CRISPR (cas) and CAS proteins, and the origin and diversity of spacer sequences, were identified and compared by BLAST. The presence of self-targeting sequences, PAMs, and the conservation of DRs were visualized using WebLogo 3.6. The CRISPR-like RNA secondary structure prediction was analyzed using RNAFold and MFold. CRISPR structures were identified in 19.6% of Pseudomonas species. In all, 113 typical CRISPR arrays with 18 putative cas were found, as were 2050 spacers, of which 52% showed homology to bacteriophages, 26% to chromosomes, and 22% to plasmids. No potential self-targeting was detected within the CRISPR array. All the found DRs can form thermodynamically stable secondary RNA structures. The comparison of the CRISPR/Cas system can help understand the environmental adaptability of each evolutionary lineage of clinically and environmentally relevant species, providing data support for bacterial typing, traceability, analysis, and exploration of unconventional CRISPR.
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Terns, Rebecca M., and Michael P. Terns. "The RNA- and DNA-targeting CRISPR–Cas immune systems of Pyrococcus furiosus." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1416–21. http://dx.doi.org/10.1042/bst20130056.
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Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5′ tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA–Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR–Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR–Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR–Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR–Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications.
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Hidalgo-Cantabrana, Claudio, and Rodolphe Barrangou. "Characterization and applications of Type I CRISPR-Cas systems." Biochemical Society Transactions 48, no. 1 (January 10, 2020): 15–23. http://dx.doi.org/10.1042/bst20190119.
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CRISPR-Cas constitutes the adaptive immune system of bacteria and archaea. This RNA-mediated sequence-specific recognition and targeting machinery has been used broadly for diverse applications in a wide range of organisms across the tree of life. The compact class 2 systems, that hinge on a single Cas effector nuclease have been harnessed for genome editing, transcriptional regulation, detection, imaging and other applications, in different research areas. However, most of the CRISPR-Cas systems belong to class 1, and the molecular machinery of the most widespread and diverse Type I systems afford tremendous opportunities for a broad range of applications. These highly abundant systems rely on a multi-protein effector complex, the CRISPR associated complex for antiviral defense (Cascade), which drives DNA targeting and cleavage. The complexity of these systems has somewhat hindered their widespread usage, but the pool of thousands of diverse Type I CRISPR-Cas systems opens new avenues for CRISPR-based applications in bacteria, archaea and eukaryotes. Here, we describe the features and mechanism of action of Type I CRISPR-Cas systems, illustrate how endogenous systems can be reprogrammed to target the host genome and perform genome editing and transcriptional regulation by co-delivering a minimal CRISPR array together with a repair template. Moreover, we discuss how these systems can also be used in eukaryotes. This review provides a framework for expanding the CRISPR toolbox, and repurposing the most abundant CRISPR-Cas systems for a wide range of applications.
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Roberts, Avery, and Rodolphe Barrangou. "Applications of CRISPR-Cas systems in lactic acid bacteria." FEMS Microbiology Reviews 44, no. 5 (May 20, 2020): 523–37. http://dx.doi.org/10.1093/femsre/fuaa016.
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ABSTRACT As a phenotypically and phylogenetically diverse group, lactic acid bacteria are found in a variety of natural environments and occupy important roles in medicine, biotechnology, food and agriculture. The widespread use of lactic acid bacteria across these industries fuels the need for new and functionally diverse strains that may be utilized as starter cultures or probiotics. Originally characterized in lactic acid bacteria, CRISPR-Cas systems and derived molecular machines can be used natively or exogenously to engineer new strains with enhanced functional attributes. Research on CRISPR-Cas biology and its applications has exploded over the past decade with studies spanning from the initial characterization of CRISPR-Cas immunity in Streptococcus thermophilus to the use of CRISPR-Cas for clinical gene therapies. Here, we discuss CRISPR-Cas classification, overview CRISPR biology and mechanism of action, and discuss current and future applications in lactic acid bacteria, opening new avenues for their industrial exploitation and manipulation of microbiomes.
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Huang, Shuo, Rui Dai, Zhiqi Zhang, Han Zhang, Meng Zhang, Zhangjun Li, Kangrui Zhao, et al. "Clustered Regularly Interspaced Short Palindromic Repeats-Based Techniques for Live-Cell Imaging and Bioanalysis." International Journal of Molecular Sciences 24, no. 17 (August 30, 2023): 13447. http://dx.doi.org/10.3390/ijms241713447.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and associated (CRISPR/Cas) systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.
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Philippe, Cécile, and Sylvain Moineau. "The endless battle between phages and CRISPR–Cas systems in Streptococcus thermophilus." Biochemistry and Cell Biology 99, no. 4 (August 2021): 397–402. http://dx.doi.org/10.1139/bcb-2020-0593.
Full textAbstract:
This review describes the contribution of basic research on phage–bacteria interactions to the understanding of CRISPR–Cas systems and their various applications. It focuses on the natural function of CRISPR–Cas systems as adaptive defense mechanisms against mobile genetic elements such as bacteriophage genomes and plasmids. Some of the advances in the characterization of the type II-A CRISPR–Cas system of Streptococcus thermophilus and Streptococcus pyogenes led to the development of the CRISPR–Cas9 genome-editing technology. We mostly discuss the 3 stages of the CRISPR–Cas system in S. thermophilus, namely the adaptation stage, which is unique to this resistance mechanism; the CRISPR RNA biogenesis; and the DNA-cutting activity in the interference stage to protect bacteria against phages. Finally, we look into applications of CRISPR–Cas in microbiology, including overcoming limitations in genome editing.
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Pan, Meichen, Matthew A. Nethery, Claudio Hidalgo-Cantabrana, and Rodolphe Barrangou. "Comprehensive Mining and Characterization of CRISPR-Cas Systems in Bifidobacterium." Microorganisms 8, no. 5 (May 12, 2020): 720. http://dx.doi.org/10.3390/microorganisms8050720.
Full textAbstract:
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated cas) systems constitute the adaptive immune system in prokaryotes, which provides resistance against bacteriophages and invasive genetic elements. The landscape of applications in bacteria and eukaryotes relies on a few Cas effector proteins that have been characterized in detail. However, there is a lack of comprehensive studies on naturally occurring CRISPR-Cas systems in beneficial bacteria, such as human gut commensal Bifidobacterium species. In this study, we mined 954 publicly available Bifidobacterium genomes and identified CRIPSR-Cas systems in 57% of these strains. A total of five CRISPR-Cas subtypes were identified as follows: Type I-E, I-C, I-G, II-A, and II-C. Among the subtypes, Type I-C was the most abundant (23%). We further characterized the CRISPR RNA (crRNA), tracrRNA, and PAM sequences to provide a molecular basis for the development of new genome editing tools for a variety of applications. Moreover, we investigated the evolutionary history of certain Bifidobacterium strains through visualization of acquired spacer sequences and demonstrated how these hypervariable CRISPR regions can be used as genotyping markers. This extensive characterization will enable the repurposing of endogenous CRISPR-Cas systems in Bifidobacteria for genome engineering, transcriptional regulation, genotyping, and screening of rare variants.
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McBride, Tess M., Shaharn C. Cameron, Peter C. Fineran, and Robert D. Fagerlund. "The biology and type I/III hybrid nature of type I-D CRISPR–Cas systems." Biochemical Journal 480, no. 7 (April 13, 2023): 471–88. http://dx.doi.org/10.1042/bcj20220073.
Full textAbstract:
Prokaryotes have adaptive defence mechanisms that protect them from mobile genetic elements and viral infection. One defence mechanism is called CRISPR–Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins). There are six different types of CRISPR–Cas systems and multiple subtypes that vary in composition and mode of action. Type I and III CRISPR–Cas systems utilise multi-protein complexes, which differ in structure, nucleic acid binding and cleaving preference. The type I-D system is a chimera of type I and III systems. Recently, there has been a burst of research on the type I-D CRISPR–Cas system. Here, we review the mechanism, evolution and biotechnological applications of the type I-D CRISPR–Cas system.
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Rybnicky, Grant A., Nicholas A. Fackler, Ashty S. Karim, Michael Köpke, and Michael C. Jewett. "Spacer2PAM: A computational framework to guide experimental determination of functional CRISPR-Cas system PAM sequences." Nucleic Acids Research 50, no. 6 (March 8, 2022): 3523–34. http://dx.doi.org/10.1093/nar/gkac142.
Full textAbstract:
Abstract RNA-guided nucleases from CRISPR-Cas systems expand opportunities for precise, targeted genome modification. Endogenous CRISPR-Cas systems in many prokaryotes are attractive to circumvent expression, functionality, and unintended activity hurdles posed by heterologous CRISPR-Cas effectors. However, each CRISPR-Cas system recognizes a unique set of protospacer adjacent motifs (PAMs), which requires identification by extensive screening of randomized DNA libraries. This challenge hinders development of endogenous CRISPR-Cas systems, especially those based on multi-protein effectors and in organisms that are slow-growing or have transformation idiosyncrasies. To address this challenge, we present Spacer2PAM, an easy-to-use, easy-to-interpret R package built to predict and guide experimental determination of functional PAM sequences for any CRISPR-Cas system given its corresponding CRISPR array as input. Spacer2PAM can be used in a ‘Quick’ method to generate a single PAM prediction or in a ‘Comprehensive’ method to inform targeted PAM libraries small enough to screen in difficult to transform organisms. We demonstrate Spacer2PAM by predicting PAM sequences for industrially relevant organisms and experimentally identifying seven PAM sequences that mediate interference from the Spacer2PAM-informed PAM library for the type I-B CRISPR-Cas system from Clostridium autoethanogenum. We anticipate that Spacer2PAM will facilitate the use of endogenous CRISPR-Cas systems for industrial biotechnology and synthetic biology.
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Arefyeva, N. A., Yu P. Dzhioev, A. Yu Borisenko, V. I. Chemerilova, O. F. Vyatchina, O. A. Sekerina, L. A. Stepanenko, et al. "BIOINFORMATIC SEARCH OF CRISPR/CAS SYSTEM STRUCTURES IN GENOME OF PCT281 PLASMID OF BACILLUS THURINGIENSIS SUBSP. CHINENSIS STRAIN CT-43." Acta Biomedica Scientifica 3, no. 5 (October 29, 2018): 33–38. http://dx.doi.org/10.29413/abs.2018-3.5.5.
Full textAbstract:
Background.CRISPR/Cas systems loci are one of the functionally important patterns in bacterial genome which perform the role of “adaptive immune defense” from foreign nucleic acids. The study of CRISPR/Cas systems structure in genomes of plasmids and phages provide new information about the evolution of this systems in bacterial hosts.Aims.A search of CRISPR/Cas systems structures in pCT281 plasmid from Bacillus thuringiensis subsp. chinensis strain CT-43 using bioinformatic methods.Materials and methods.Search studies using bioinformatics methods were performed with the genome of pCT281 plasmid of B. thuringiensis subsp. chinensis strain CT-43 from the RefSeq database. To search for the CRISPR/Cas system structure MacSyFinder (ver. 1.0.5) and three combined algorithms were used: CRISPRFinder; PILER-CR; CRISPR Recognition Tool (CRT). The consensus repeat sequence was generated in WebLogo 3.Results and discussion.In pCT281 plasmid we detected one locus of CRISPR/Cas system of the type I-C which contains 2 CRISPR-cassettes and 4 cas-genes located between them. The CRISPR-cassette 1 includes 10 spacers from 32 to 35 bp and 11 repeats 32bp in length. 5 spacers (33–35 bp) separated by 6 repeats 32 bp in length were detected in the CRISPR-cassette 2.Conclusions.The bioinformatic methods used in this study enable to conduct a search of CRISPR/Cas systems structures in plasmid genomes. The presence of the CRISPR-Cas locus in pCT281 plasmid confirms a possible transfer of this system from the nucleoid to this plasmid. The detected spacers provide information about phages this bacteria was encountered.
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Peretolchina, N. P., Yu P. Dzhioev, A. Yu Borisenko, L. A. Stepanenko, E. A. Voskresenskaya, V. T. Klimov, O. N. Reva, and V. I. Zlobin. "In silico comparative analysis of crispr-cas system structures of Yersinia pseudotuberculosis causing different clinical manifestations of pseudotuberculosis." Journal Infectology 11, no. 2 (May 17, 2019): 80–87. http://dx.doi.org/10.22625/2072-6732-2019-11-2-80-87.
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The aim of this research was to analyze and compare CRIPSR loci and cas-proteins of Yersinia pseudotuberculosis strains isolated in different territories from patients with various clinical manifestations of pseudotuberculosis.Materials and Methods. Complete genomes of Y. pseudotuberculosis IP329353 (NC_006155) and IP31758 (NC_009708) were obtained from NCBI Nucleotide Database. Strains were isolated from patients with gastroenteritis and systemic infection respectively. Search, identification, and analysis of CRISPR systems were carried out by onlinetools CRISPROne, CRISPRDetect, and CRISPRTarget.Results. Analyzed strains have CRISPR-Cas systems that include one set of cas-genes and arrays situated at the long distances from each other. We defined three CRISPR arrays in Y. pseudotuberculosis IP32953: array YP1 located near cas-genes, arrays YP2 and YP3. CRISPR-Cas system of Y. pseudotuberculosis IP31758 includes two arrays – YP1 and YP3. CRISPR systems do not share similar spacers.Conclusion. CRISPR systems of the analyzed strains differ in CRISPR loci and cas-protein structures that can be used as specific molecular marks of analyzed strains during the study of intra-species variability and evolution of Y. pseudotuberculosis.
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Parkes, Ashley, Fiona Kemm, Liu He, and Tom Killelea. "CRISPR systems: what’s new, where next?" Biochemist 43, no. 6 (December 15, 2021): 46–50. http://dx.doi.org/10.1042/bio_2021_194.
Full textAbstract:
The genetic signature of natural CRISPR-Cas systems were first noted in a 1989 publication and were characterized in detail from 2002 to 2007, culminating in the first report of a prokaryotic adaptive immune system. Since then, CRISPR-Cas enzymes have been adapted into molecular biology tools that have transformed genetic engineering across domains of life. In this feature article, we describe origins, uses and futures of CRISPR-Cas enzymes in genetic engineering: we highlight advances made in the past 10 years. Central to these advances is appreciation of interplay between CRISPR engineering and DNA repair. We highlight how this relationship has been manipulated to create further advances in the development of gene editing.
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Alkhnbashi, Omer S., Alexander Mitrofanov, Robson Bonidia, Martin Raden, Van Dinh Tran, Florian Eggenhofer, Shiraz A. Shah, et al. "CRISPRloci: comprehensive and accurate annotation of CRISPR–Cas systems." Nucleic Acids Research 49, W1 (June 16, 2021): W125—W130. http://dx.doi.org/10.1093/nar/gkab456.
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Abstract CRISPR–Cas systems are adaptive immune systems in prokaryotes, providing resistance against invading viruses and plasmids. The identification of CRISPR loci is currently a non-standardized, ambiguous process, requiring the manual combination of multiple tools, where existing tools detect only parts of the CRISPR-systems, and lack quality control, annotation and assessment capabilities of the detected CRISPR loci. Our CRISPRloci server provides the first resource for the prediction and assessment of all possible CRISPR loci. The server integrates a series of advanced Machine Learning tools within a seamless web interface featuring: (i) prediction of all CRISPR arrays in the correct orientation; (ii) definition of CRISPR leaders for each locus; and (iii) annotation of cas genes and their unambiguous classification. As a result, CRISPRloci is able to accurately determine the CRISPR array and associated information, such as: the Cas subtypes; cassette boundaries; accuracy of the repeat structure, orientation and leader sequence; virus-host interactions; self-targeting; as well as the annotation of cas genes, all of which have been missing from existing tools. This annotation is presented in an interactive interface, making it easy for scientists to gain an overview of the CRISPR system in their organism of interest. Predictions are also rendered in GFF format, enabling in-depth genome browser inspection. In summary, CRISPRloci constitutes a full suite for CRISPR–Cas system characterization that offers annotation quality previously available only after manual inspection.
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Borisenko, A. Yu, N. A. Arefieva, Yu P. Dzhioev, S. V. Erdyneev, Yu S. Bukin, G. A. Teterina, A. A. Pristavka, et al. "In Silico Analysis of the Structural Diversity of CRISPR-Cas Systems in Genomes of Salmonella enterica and Phage Species Detected by Them." Bulletin of Irkutsk State University. Series Biology. Ecology 45 (2023): 3–20. http://dx.doi.org/10.26516/2073-3372.2023.45.3.
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The problem of resistance of pathogenic bacteria to antibiotics has become global and, therefore, there is renewed interest in the use of bacteriophages. However, bacteria also have phage defense structures, the CRISPR/Cas system. Therefore, the analysis of the structural diversity of CRISPR-Cas systems in the genomes of pathogenic bacteria and phages is an important fundamental and applied direction. The aim. Investigation of the diversity of structures of CRISPR/Cas systems in the genomes of S. enterica strains from the NCBI database using bioinformatics programs and assessment of the possibilities to identify phage protection of strains through spacers in CRISPR cassettes. The studies were carried out with the genomes of 449 S. enterica strains from the NCBI database. A number of bioinformation software methods were used: 1) MacSyFinder, 2) CRISPR Interactive database, 3) CRISPR R Tool, 4) CRISPI: a CRISPR Interactive database, 5) CRISPRFinder. Screening of phages through spacers CRISPR cassettes was used: 1) CRISPRTarget, 2) Mycobacteriophage Database, 3) Phages database. In the genomes of the studied strains of S. enterica, one type of CRISPR/Cas system, I-E, was identified. Protein genes were present in each locus of the CRISPR/Cas systems: Cas1_0_I-E_7, Cas2_0_I-E_8, Cas3_0_I_1, Cas5_0_I-E_5, Cas6_0_I-E_6, Cas7_0_I-E_4, Cse1_0_I-E_2, Cse2_0_I-E_3. The number of cassettes was from 1 to 3, and the spacers in them varied from 8 to 30. Repeats in CRISPR cassettes varied from 27 to 29 base pairs. The identified phages belonged to bacteria of the genera: Salmonella – 60%, Escherichia – 18%, Enterobacter – 9%, Salmonella – 8%, and Staphylococcus and Enterococcus were up to 5%. The obtained data on the diversity of CRISPR/Cas systems in the genomes of the studied S. enterica strains demonstrate their unique structures. The homogeneity of CRISPR/Cas systems and the rooting of CAS types I-E in genomes can be explained by their participation in the interspecific transmission of these CRISPR systems.
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Lin, Weijia. "Application of CRISPR-Cas System in the Treatment of Human Viral Disease." BIO Web of Conferences 59 (2023): 02003. http://dx.doi.org/10.1051/bioconf/20235902003.
Full textAbstract:
CRISPR-Cas systems, consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas), are the latest generation of gene editing technology and have been widely used in molecular biology research. CRISPR-Cas systems also have unlimited potential in the field of medicine, especially in the treatment of human viral diseases, such as blocking virus invasion, interfering with virus replication, and eliminating viral genome and sequelae of virus infection. In this article, the latest research progress of CRISPR-Cas9 system and other CRISPR systems in treatments of several viral diseases are reviewed. In addition, the advantages and potential problems of CRISPR systems as treatment options are analyzed to provide ideas for subsequent related research.
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Zhu, Yuwei, and Zhiwei Huang. "Recent advances in structural studies of the CRISPR-Cas-mediated genome editing tools." National Science Review 6, no. 3 (November 29, 2018): 438–51. http://dx.doi.org/10.1093/nsr/nwy150.
Full textAbstract:
Abstract Clustered regularly interspaced short palindromic repeats (CRISPR) and accompanying CRISPR-associated (Cas) proteins provide RNA-guided adaptive immunity for prokaryotes to defend themselves against viruses. The CRISPR-Cas systems have attracted much attention in recent years for their power in aiding the development of genome editing tools. Based on the composition of the CRISPR RNA-effector complex, the CRISPR-Cas systems can be divided into two classes and six types. In this review, we summarize recent advances in the structural biology of the CRISPR-Cas-mediated genome editing tools, which helps us to understand the mechanism of how the guide RNAs assemble with diverse Cas proteins to cleave target nucleic acids.
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Brezgin, Sergey, Anastasiya Kostyusheva, Dmitry Kostyushev, and Vladimir Chulanov. "Dead Cas Systems: Types, Principles, and Applications." International Journal of Molecular Sciences 20, no. 23 (November 30, 2019): 6041. http://dx.doi.org/10.3390/ijms20236041.
Full textAbstract:
The gene editing tool CRISPR-Cas has become the foundation for developing numerous molecular systems used in research and, increasingly, in medical practice. In particular, Cas proteins devoid of nucleolytic activity (dead Cas proteins; dCas) can be used to deliver functional cargo to programmed sites in the genome. In this review, we describe current CRISPR systems used for developing different dCas-based molecular approaches and summarize their most significant applications. We conclude with comments on the state-of-art in the CRISPR field and future directions.
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Tyumentseva, Marina, Yulia Mikhaylova, Anna Prelovskaya, Konstantin Karbyshev, Aleksandr Tyumentsev, Lyudmila Petrova, Anna Mironova, Mikhail Zamyatin, Andrey Shelenkov, and Vasiliy Akimkin. "CRISPR Element Patterns vs. Pathoadaptability of Clinical Pseudomonas aeruginosa Isolates from a Medical Center in Moscow, Russia." Antibiotics 10, no. 11 (October 26, 2021): 1301. http://dx.doi.org/10.3390/antibiotics10111301.
Full textAbstract:
Pseudomonas aeruginosa is a member of the ESKAPE opportunistic pathogen group, which includes six species of the most dangerous microbes. This pathogen is characterized by the rapid acquisition of antimicrobial resistance, thus causing major healthcare concerns. This study presents a comprehensive analysis of clinical P. aeruginosa isolates based on whole-genome sequencing data. The isolate collection studied was characterized by a variety of clonal lineages with a domination of high-risk epidemic clones and different CRISPR/Cas element patterns. This is the first report on the coexistence of two and even three different types of CRISPR/Cas systems simultaneously in Russian clinical strains of P. aeruginosa. The data include molecular typing and genotypic antibiotic resistance determination, as well as the phylogenetic analysis of the full-length cas gene and anti-CRISPR genes sequences, predicted prophage sequences, and conducted a detailed CRISPR array analysis. The differences between the isolates carrying different types and quantities of CRISPR/Cas systems were investigated. The pattern of virulence factors in P. aeruginosa isolates lacking putative CRISPR/Cas systems significantly differed from that of samples with single or multiple putative CRISPR/Cas systems. We found significant correlations between the numbers of prophage sequences, antibiotic resistance genes, and virulence genes in P. aeruginosa isolates with different patterns of CRISPR/Cas-elements. We believe that the data presented will contribute to further investigations in the field of bacterial pathoadaptability, including antimicrobial resistance and the role of CRISPR/Cas systems in the plasticity of the P. aeruginosa genome.
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Høyland-Kroghsbo, Nina M., Jon Paczkowski, Sampriti Mukherjee, Jenny Broniewski, Edze Westra, Joseph Bondy-Denomy, and Bonnie L. Bassler. "Quorum sensing controls thePseudomonas aeruginosaCRISPR-Cas adaptive immune system." Proceedings of the National Academy of Sciences 114, no. 1 (November 14, 2016): 131–35. http://dx.doi.org/10.1073/pnas.1617415113.
Full textAbstract:
CRISPR-Cas are prokaryotic adaptive immune systems that provide protection against bacteriophage (phage) and other parasites. Little is known about how CRISPR-Cas systems are regulated, preventing prediction of phage dynamics in nature and manipulation of phage resistance in clinical settings. Here, we show that the bacteriumPseudomonas aeruginosaPA14 uses the cell–cell communication process, called quorum sensing, to activatecasgene expression, to increase CRISPR-Cas targeting of foreign DNA, and to promote CRISPR adaptation, all at high cell density. This regulatory mechanism ensures maximum CRISPR-Cas function when bacterial populations are at highest risk for phage infection. We demonstrate that CRISPR-Cas activity and acquisition of resistance can be modulated by administration of pro- and antiquorum-sensing compounds. We propose that quorum-sensing inhibitors could be used to suppress the CRISPR-Cas adaptive immune system to enhance medical applications, including phage therapies.
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Peters, Joseph E., Kira S. Makarova, Sergey Shmakov, and Eugene V. Koonin. "Recruitment of CRISPR-Cas systems by Tn7-like transposons." Proceedings of the National Academy of Sciences 114, no. 35 (August 15, 2017): E7358—E7366. http://dx.doi.org/10.1073/pnas.1709035114.
Full textAbstract:
A survey of bacterial and archaeal genomes shows that many Tn7-like transposons contain minimal type I-F CRISPR-Cas systems that consist of fused cas8f and cas5f, cas7f, and cas6f genes and a short CRISPR array. Several small groups of Tn7-like transposons encompass similarly truncated type I-B CRISPR-Cas. This minimal gene complement of the transposon-associated CRISPR-Cas systems implies that they are competent for pre-CRISPR RNA (precrRNA) processing yielding mature crRNAs and target binding but not target cleavage that is required for interference. Phylogenetic analysis demonstrates that evolution of the CRISPR-Cas–containing transposons included a single, ancestral capture of a type I-F locus and two independent instances of type I-B loci capture. We show that the transposon-associated CRISPR arrays contain spacers homologous to plasmid and temperate phage sequences and, in some cases, chromosomal sequences adjacent to the transposon. We hypothesize that the transposon-encoded CRISPR-Cas systems generate displacement (R-loops) in the cognate DNA sites, targeting the transposon to these sites and thus facilitating their spread via plasmids and phages. These findings suggest the existence of RNA-guided transposition and fit the guns-for-hire concept whereby mobile genetic elements capture host defense systems and repurpose them for different stages in the life cycle of the element.
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Bernheim, Aude, David Bikard, Marie Touchon, and Eduardo P. C. Rocha. "A matter of background: DNA repair pathways as a possible cause for the sparse distribution of CRISPR-Cas systems in bacteria." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (March 25, 2019): 20180088. http://dx.doi.org/10.1098/rstb.2018.0088.
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The absence of CRISPR-Cas systems in more than half of the sequenced bacterial genomes is intriguing, because their role in adaptive immunity and their frequent transfer between species should have made them almost ubiquitous, as is the case in Archaea. Here, we investigate the possibility that the success of CRISPR-Cas acquisition by horizontal gene transfer is affected by the interactions of these systems with the host genetic background and especially with components of double-strand break repair systems (DSB-RS). We first described the distribution of systems specialized in the repair of double-strand breaks in Bacteria: homologous recombination and non-homologous end joining. This allowed us to show that such systems are more often positively or negatively correlated with the frequency of CRISPR-Cas systems than random genes of similar frequency. The detailed analysis of these co-occurrence patterns shows that our method identifies previously known cases of mechanistic interactions between these systems. It also reveals other positive and negative patterns of co-occurrence between DSB-RS and CRISPR-Cas systems. Notably, it shows that the patterns of distribution of CRISPR-Cas systems in Proteobacteria are strongly dependent on the epistatic groups including RecBCD and AddAB. Our results suggest that the genetic background plays an important role in the success of adaptive immunity in different bacterial clades and provide insights to guide further experimental research on the interactions between CRISPR-Cas and DSB-RS. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Heussler, Gary E., and George A. O'Toole. "Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems." Journal of Bacteriology 198, no. 10 (February 29, 2016): 1481–86. http://dx.doi.org/10.1128/jb.00086-16.
Full textAbstract:
Clusteredregularlyinterspacedshortpalindromicrepeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPR-Cas self-targeting is vital to fully understanding the spectrum of function of these systems.
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Tyumentseva, Marina, Yulia Mikhaylova, Anna Prelovskaya, Aleksandr Tyumentsev, Lyudmila Petrova, Valeria Fomina, Mikhail Zamyatin, Andrey Shelenkov, and Vasiliy Akimkin. "Genomic and Phenotypic Analysis of Multidrug-Resistant Acinetobacter baumannii Clinical Isolates Carrying Different Types of CRISPR/Cas Systems." Pathogens 10, no. 2 (February 13, 2021): 205. http://dx.doi.org/10.3390/pathogens10020205.
Full textAbstract:
Acinetobacter baumannii is an opportunistic pathogen being one of the most important causative agents of a wide range of nosocomial infections associated with multidrug resistance and high mortality rate. This study presents a multiparametric and correlation analyses of clinical multidrug-resistant A. baumannii isolates using short- and long-read whole-genome sequencing, which allowed us to reveal specific characteristics of the isolates with different CRISPR/Cas systems. We also compared antibiotic resistance and virulence gene acquisition for the groups of the isolates having functional CRISPR/Cas systems, just CRISPR arrays without cas genes, and without detectable CRISPR spacers. The data include three schemes of molecular typing, phenotypic and genotypic antibiotic resistance determination, as well as phylogenetic analysis of full-length cas gene sequences, predicted prophage sequences and CRISPR array type determination. For the first time the differences between the isolates carrying Type I-F1 and Type I-F2 CRISPR/Cas systems were investigated. A. baumannii isolates with Type I-F1 system were shown to have smaller number of reliably detected CRISPR arrays, and thus they could more easily adapt to environmental conditions through acquisition of antibiotic resistance genes, while Type I-F2 A. baumannii might have stronger “immunity” and use CRISPR/Cas system to block the dissemination of these genes. In addition, virulence factors abaI, abaR, bap and bauA were overrepresented in A. baumannii isolates lacking CRISPR/Cas system. This indicates the role of CRISPR/Cas in fighting against phage infections and preventing horizontal gene transfer. We believe that the data presented will contribute to further investigations in the field of antimicrobial resistance and CRISPR/Cas studies.
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Stevanovic, Marta, Elena Piotter, Michelle E. McClements, and Robert E. MacLaren. "CRISPR Systems Suitable for Single AAV Vector Delivery." Current Gene Therapy 22, no. 1 (February 2022): 1–14. http://dx.doi.org/10.2174/1566523221666211006120355.
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Abstract: CRISPR (clustered regularly interspaced short palindromic repeats)/Cas gene editing is a revolutionary technology that can enable the correction of genetic mutations in vivo, providing great promise as a therapeutic intervention for inherited diseases. Adeno-associated viral (AAV) vectors are a potential vehicle for delivering CRISPR/Cas. However, they are restricted by their limited packaging capacity. Identifying smaller Cas orthologs that can be packaged, along with the required guide RNA elements, into a single AAV would be an important optimization for CRISPR/- Cas gene editing. Expanding the options of Cas proteins that can be delivered by a single AAV not only increases translational application but also expands the genetic sites that can be targeted for editing. This review considers the benefits and current scope of small Cas protein orthologs that are suitable for gene editing approaches using single AAV vector delivery.
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Mlaga, Kodjovi D., Vincent Garcia, Philippe Colson, Raymond Ruimy, Jean-Marc Rolain, and Seydina M. Diene. "Extensive Comparative Genomic Analysis of Enterococcus faecalis and Enterococcus faecium Reveals a Direct Association between the Absence of CRISPR–Cas Systems, the Presence of Anti-Endonuclease (ardA) and the Acquisition of Vancomycin Resistance in E. faecium." Microorganisms 9, no. 6 (May 21, 2021): 1118. http://dx.doi.org/10.3390/microorganisms9061118.
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Here, we performed a comparative genomic analysis of all available genomes of E. faecalis (n = 1591) and E. faecium (n = 1981) and investigated the association between the presence or absence of CRISPR-Cas systems, endonuclease/anti-endonuclease systems and the acquisition of antimicrobial resistance, especially vancomycin resistance genes. Most of the analysed Enterococci were isolated from humans and less than 14% of them were from foods and animals. We analysed and detected CRISPR–Cas systems in 75.36% of E. faecalis genomes and only 4.89% of E. faecium genomes with a significant difference (p-value < 10−5). We found a negative correlation between the number of CRISPR–Cas systems and genome size (r = −0.397, p-value < 10−5) and a positive correlation between the genome %GC content and the number of CRISPR–Cas systems (r = 0.215, p-value < 10−5). Our findings showed that the presence of the anti-endonuclease ardA gene may explain the decrease in the number of CRISPR–Cas systems in E. faecium, known to deactivate the endonucleases’ protective activities and enable the E. faecium genome to be versatile in acquiring mobile genetic elements, including carriers of antimicrobial resistance genes, especially vanB. Most importantly, we observed that there was a direct association between the absence of CRISPR–Cas, the presence of the anti-CRISPR ardA gene and the acquisition of vancomycin resistance genes.
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Wang, Chuan, Yuze Yang, Shaoqing Tang, Yuanzi Liu, Yaqin Wei, Xuerui Wan, Yajuan Liu, Zhao Zhang, and Yongjie Sunkang. "Comparison of Structural Features of CRISPR-Cas Systems in Thermophilic Bacteria." Microorganisms 11, no. 9 (September 10, 2023): 2275. http://dx.doi.org/10.3390/microorganisms11092275.
Full textAbstract:
The clustered regularly interspaced short palindromic repeat (CRISPR) is an adaptive immune system that defends most archaea and many bacteria from foreign DNA, such as phages, viruses, and plasmids. The link between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria remains unclear. To investigate the relationship between the structural characteristics, diversity, and distribution properties of the CRISPR-Cas system and the optimum growth temperature in thermophilic bacteria, genomes of 61 species of thermophilic bacteria with complete genome sequences were downloaded from GenBank in this study. We used CRISPRFinder to extensively study CRISPR structures and CRISPR-associated genes (cas) from thermophilic bacteria. We statistically analyzed the association between the CRISPR-Cas system and the optimum growth temperature of thermophilic bacteria. The results revealed that 59 strains of 61 thermophilic bacteria had at least one CRISPR locus, accounting for 96.72% of the total. Additionally, a total of 362 CRISPR loci, 209 entirely distinct repetitive sequences, 131 cas genes, and 7744 spacer sequences were discovered. The average number of CRISPR loci and the average minimum free energy (MFE) of the RNA secondary structure of repeat sequences were positively correlated with temperature whereas the average length of CRISPR loci and the average number of spacers were negatively correlated. The temperature did not affect the average number of CRISPR loci, the average length of repeats, or the guanine-cytosine (GC) content of repeats. The average number of CRISPR loci, the average length of the repeats, and the GC content of the repeats did not reflect temperature dependence. This study may provide a new basis for the study of the thermophilic bacterial adaptation mechanisms of thermophilic bacteria.
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Liu, Tina Y., and Jennifer A. Doudna. "Chemistry of Class 1 CRISPR-Cas effectors: Binding, editing, and regulation." Journal of Biological Chemistry 295, no. 42 (August 14, 2020): 14473–87. http://dx.doi.org/10.1074/jbc.rev120.007034.
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Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.
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Kostyushev, Dmitry, Anastasiya Kostyusheva, Sergey Brezgin, Valery Smirnov, Elena Volchkova, Alexander Lukashev, and Vladimir Chulanov. "Gene Editing by Extracellular Vesicles." International Journal of Molecular Sciences 21, no. 19 (October 5, 2020): 7362. http://dx.doi.org/10.3390/ijms21197362.
Full textAbstract:
CRISPR/Cas technologies have advanced dramatically in recent years. Many different systems with new properties have been characterized and a plethora of hybrid CRISPR/Cas systems able to modify the epigenome, regulate transcription, and correct mutations in DNA and RNA have been devised. However, practical application of CRISPR/Cas systems is severely limited by the lack of effective delivery tools. In this review, recent advances in developing vehicles for the delivery of CRISPR/Cas in the form of ribonucleoprotein complexes are outlined. Most importantly, we emphasize the use of extracellular vesicles (EVs) for CRISPR/Cas delivery and describe their unique properties: biocompatibility, safety, capacity for rational design, and ability to cross biological barriers. Available molecular tools that enable loading of desired protein and/or RNA cargo into the vesicles in a controllable manner and shape the surface of EVs for targeted delivery into specific tissues (e.g., using targeting ligands, peptides, or nanobodies) are discussed. Opportunities for both endogenous (intracellular production of CRISPR/Cas) and exogenous (post-production) loading of EVs are presented.
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Ramachandran, Gayetri, and David Bikard. "Editing the microbiome the CRISPR way." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (March 25, 2019): 20180103. http://dx.doi.org/10.1098/rstb.2018.0103.
Full textAbstract:
Our bodies are colonized by a complex ecosystem of bacteria, unicellular eukaryotes and their viruses that together play a major role in our health. Over the past few years tools derived from the prokaryotic immune system known as CRISPR-Cas have empowered researchers to modify and study organisms with unprecedented ease and efficiency. Here we discuss how various types of CRISPR-Cas systems can be used to modify the genome of gut microorganisms and bacteriophages. CRISPR-Cas systems can also be delivered to bacterial population and programmed to specifically eliminate members of the microbiome. Finally, engineered CRISPR-Cas systems can be used to control gene expression and modulate the production of metabolites and proteins. Together these tools provide exciting opportunities to investigate the complex interplay between members of the microbiome and our bodies, and present new avenues for the development of drugs that target the microbiome. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Fallah, Mahnaz Shafaei, Alireza Mohebbi, Mohammad Yasaghi, and Ezzat Allah Ghaemi. "CRISPR-Cas systems in Proteus mirabilis." Infection, Genetics and Evolution 92 (August 2021): 104881. http://dx.doi.org/10.1016/j.meegid.2021.104881.
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