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1
Huescas, C. G. Y., R. I. Pereira, J. Prichula, P. A. Azevedo, J. Frazzon, and A. P. G. Frazzon. "Frequency of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) in non-clinical Enterococcus faecalis and Enterococcus faecium strains." Brazilian Journal of Biology 79, no. 3 (September 2019): 460–65. http://dx.doi.org/10.1590/1519-6984.183375.
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Abstract The fidelity of the genomes is defended by mechanism known as Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) systems. Three Type II CRISPR systems (CRISPR1- cas, CRISPR2 and CRISPR3-cas) have been identified in enterococci isolates from clinical and environmental samples. The aim of this study was to observe the distribution of CRISPR1-cas, CRISPR2 and CRISPR3-cas in non-clinical strains of Enterococcus faecalis and Enterococcus faecium isolates from food and fecal samples, including wild marine animals. The presence of CRISPRs was evaluated by PCR in 120 enterococci strains, 67 E. faecalis and 53 E. faecium. It is the first report of the presence of the CRISPRs system in E. faecalis and E. faecium strains isolated from wild marine animal fecal samples. The results showed that in non-clinical strains, the CRISPRs were more frequently detected in E. faecalis than in E. faecium. And the frequencies of CRISPR1-cas and CRISPR2 were higher (60%) in E. faecalis strains isolated from animal feces, compared to food samples. Both strains showed low frequencies of CRISPR3-cas (8.95% and 1.88%). In conclusion, the differences in the habitats of enterococcal species may be related with the results observe in distribution of CRISPRs systems.
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Serbanescu, M. A., M. Cordova, K. Krastel, R. Flick, N. Beloglazova, A. Latos, A. F. Yakunin, D. B. Senadheera, and D. G. Cvitkovitch. "Role of the Streptococcus mutans CRISPR-Cas Systems in Immunity and Cell Physiology." Journal of Bacteriology 197, no. 4 (December 8, 2014): 749–61. http://dx.doi.org/10.1128/jb.02333-14.
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CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacteriumStreptococcus mutansUA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of otherS. mutans. The deletion of thecasgenes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) inS. mutansUA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation ofS. mutansby the plasmids matching the spacers 2 and 3. Functional analysis of thecasdeletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology inS. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression ofcasgenes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we providein vivoevidence that the type II-A CRISPR-Cas system ofS. mutansmay be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.
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Chapman, Brittany, Jeong Hoon Han, Hong Jo Lee, Isabella Ruud, and Tae Hyun Kim. "Targeted Modulation of Chicken Genes In Vitro Using CRISPRa and CRISPRi Toolkit." Genes 14, no. 4 (April 13, 2023): 906. http://dx.doi.org/10.3390/genes14040906.
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Engineering of clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated protein 9 (Cas9) system has enabled versatile applications of CRISPR beyond targeted DNA cleavage. Combination of nuclease-deactivated Cas9 (dCas9) and transcriptional effector domains allows activation (CRISPRa) or repression (CRISPRi) of target loci. To demonstrate the effectiveness of the CRISPR-mediated transcriptional regulation in chickens, three CRISPRa (VP64, VPR, and p300) and three CRISPRi (dCas9, dCas9-KRAB, and dCas9-KRAB-MeCP2) systems were tested in chicken DF-1 cells. By introducing guide RNAs (gRNAs) targeting near the transcription start site (TSS) of each gene in CRISPRa and CRISPRi effector domain-expressing chicken DF-1 cell lines, significant gene upregulation was induced in dCas9-VPR and dCas9-VP64 cells, while significant downregulation was observed with dCas9 and dCas9-KRAB. We further investigated the effect of gRNA positions across TSS and discovered that the location of gRNA is an important factor for targeted gene regulation. RNA sequencing analysis of IRF7 CRISPRa and CRISPRi- DF-1 cells revealed the specificity of CRISPRa and CRISPRi-based targeted transcriptional regulation with minimal off-target effects. These findings suggest that the CRISPRa and CRISPRi toolkits are an effective and adaptable platform for studying the chicken genome by targeted transcriptional modulation.
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La Russa, Marie F., and Lei S. Qi. "The New State of the Art: Cas9 for Gene Activation and Repression." Molecular and Cellular Biology 35, no. 22 (September 14, 2015): 3800–3809. http://dx.doi.org/10.1128/mcb.00512-15.
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CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.
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Karlson, Chou Khai Soong, Siti Nurfadhlina Mohd-Noor, Nadja Nolte, and Boon Chin Tan. "CRISPR/dCas9-Based Systems: Mechanisms and Applications in Plant Sciences." Plants 10, no. 10 (September 29, 2021): 2055. http://dx.doi.org/10.3390/plants10102055.
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RNA-guided genomic transcriptional regulation tools, namely clustered regularly interspaced short palindromic repeats interference (CRISPRi) and CRISPR-mediated gene activation (CRISPRa), are a powerful technology for gene functional studies. Deriving from the CRISPR/Cas9 system, both systems consist of a catalytically dead Cas9 (dCas9), a transcriptional effector and a single guide RNA (sgRNA). This type of dCas9 is incapable to cleave DNA but retains its ability to specifically bind to DNA. The binding of the dCas9/sgRNA complex to a target gene results in transcriptional interference. The CRISPR/dCas9 system has been explored as a tool for transcriptional modulation and genome imaging. Despite its potential applications and benefits, the challenges and limitations faced by the CRISPR/dCas9 system include the off-target effects, protospacer adjacent motif (PAM) sequence requirements, efficient delivery methods and the CRISPR/dCas9-interfered crops being labeled as genetically modified organisms in several countries. This review highlights the progression of CRISPR/dCas9 technology as well as its applications and potential challenges in crop improvement.
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Yang, Jiayi. "Applications of the CRISPR-Cas9 system in cancer models." Theoretical and Natural Science 21, no. 1 (December 20, 2023): 28–33. http://dx.doi.org/10.54254/2753-8818/21/20230804.
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Cancer has a high mortality and prevalence rate in the world. CRISPR-Cas9 is one of the novel and most common gene-editing techniques. Compared with the first two generations of gene-editing technologies, CRISPR-Cas9 system has the advantages of easy design, low cost, high efficiency and so on. sgRNA guides Cas9 to the site of the targeted gene, and Cas9 cuts the DNA strand at that site, triggering the NHEJ or HDR mechanism so as to achieve the purpose of deletion or insertion. CRISPR-Cas9 can be combined with other factors for other purposes, such as CRISPRa, CRISPRi, and base editing. The CRISPR system now has been used extensively for research into biological mechanisms and disease treatments. Since cancer is controlled by genes, a number of researchers in recent years have looked at using the CRISPR system to treat cancer. The CRISPR technology has greatly improved our understanding of cancer and the factors that affect it, and has had a major impact on the study and treatment of cancer. CRISPR gene editing can quickly and efficiently generate gene knockouts and regulate gene expression to identify relevant genes that influence cancer growth. This review systematically introduces CRISPR-Cas9 and its application methods, delivery modes, and discusses some studies using cell lines and organoids in vitro and animal models for cancer therapy in vivo.
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Shi, Yuqian. "CRISPR/Cas System in Human Genetic Diseases." Highlights in Science, Engineering and Technology 74 (December 29, 2023): 78–85. http://dx.doi.org/10.54097/ztchmw71.
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Clustered regularly interspaced short palindromic repeats/CRISPR-associated CRISPER/Cas system, as the current most popular gene-editing technology, shows great advantages of simple composition, good specificity and high cutting efficiency compared with other gene editing technology. With the rapid development of CRISPR-Cas systems, such as Cas9, Cas12a and Cas12f, can be used to edit the DNA of eukaryotic cells, and then successively found that Cas13a, Cas13b and Cas13d are targeted to the RNA merons. Through various modifications, scientists also developed a new type of the CRISPR-Cas system. With higher DNA-cutting activity, greater specificity, and smaller size than the natural CRISPR system, these engineered gene-editing systems form a powerful tool set for DNA sequence knockout, replacement, epigenetic editing, and even the activation and suppression of gene expression. Despite the potential problems in the practical application of CRISPR technology to be solved, it is believed that with further improvement, the CRISPR treatment technology will play a more important role in the prevention and treatment of human diseases, more perfectly and precisely. This review introduces the structure, functional mechanism and application of CRISPR/Cas system in human genetic diseases, and the current status and development of CRISPR/Cas system are summarized and prospected.
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Kiro, Ruth, Moran G. Goren, Ido Yosef, and Udi Qimron. "CRISPR adaptation in Escherichia coli subtypeI-E system." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1412–15. http://dx.doi.org/10.1042/bst20130109.
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The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called ‘spacers’, which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed ‘adaptation’. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR–Cas system of Escherichia coli.
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Heussler, Gary E., Jon L. Miller, Courtney E. Price, Alan J. Collins, and George A. O'Toole. "Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay." Journal of Bacteriology 198, no. 22 (August 29, 2016): 3080–90. http://dx.doi.org/10.1128/jb.00458-16.
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ABSTRACTCRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression ofcasgenes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich forPseudomonas aeruginosastrains with new spacer acquisition. We used this assay to demonstrate thatP. aeruginosarapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for theP. aeruginosatype I-F system, thecas1gene is required for CRISPR adaptation,recGcontributes to (but is not required for) primed CRISPR adaptation,recDis dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.IMPORTANCEOur understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants ofEscherichia coli, orcas1-cas2overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allowsP. aeruginosato rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system ofP. aeruginosa. Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that manyP. aeruginosastrains maintain a CRISPR-Cas system.
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Sasaki, Shigenori, Hirohito Ogawa, Hirokazu Katoh, and Tomoyuki Honda. "Suppression of Borna Disease Virus Replication during Its Persistent Infection Using the CRISPR/Cas13b System." International Journal of Molecular Sciences 25, no. 6 (March 20, 2024): 3523. http://dx.doi.org/10.3390/ijms25063523.
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Borna disease virus (BoDV-1) is a bornavirus that infects the central nervous systems of various animal species, including humans, and causes fatal encephalitis. BoDV-1 also establishes persistent infection in neuronal cells and causes neurobehavioral abnormalities. Once neuronal cells or normal neural networks are lost by BoDV-1 infection, it is difficult to regenerate damaged neural networks. Therefore, the development of efficient anti-BoDV-1 treatments is important to improve the outcomes of the infection. Recently, one of the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) systems, CRISPR/Cas13, has been utilized as antiviral tools. However, it is still unrevealed whether the CRISPR/Cas13 system can suppress RNA viruses in persistently infected cells. In this study, we addressed this question using persistently BoDV-1-infected cells. The CRISPR/Cas13 system targeting viral mRNAs efficiently decreased the levels of target viral mRNAs and genomic RNA (gRNA) in persistently infected cells. Furthermore, the CRISPR/Cas13 system targeting viral mRNAs also suppressed BoDV-1 infection if the system was introduced prior to the infection. Collectively, we demonstrated that the CRISPR/Cas13 system can suppress BoDV-1 in both acute and persistent infections. Our findings will open the avenue to treat prolonged infection with RNA viruses using the CRISPR/Cas13 system.
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Ding, Xiao, Lu Yu, Luo Chen, Yujie Li, Jinlun Zhang, Hanyan Sheng, Zhengwei Ren, et al. "Recent Progress and Future Prospect of CRISPR/Cas-Derived Transcription Activation (CRISPRa) System in Plants." Cells 11, no. 19 (September 28, 2022): 3045. http://dx.doi.org/10.3390/cells11193045.
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Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed.
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Barrangou, Rodolphe, Anne-Claire Coûté-Monvoisin, Buffy Stahl, Isabelle Chavichvily, Florian Damange, Dennis A. Romero, Patrick Boyaval, Christophe Fremaux, and Philippe Horvath. "Genomic impact of CRISPR immunization against bacteriophages." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1383–91. http://dx.doi.org/10.1042/bst20130160.
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CRISPR (clustered regularly interspaced short palindromic repeats) together with cas (CRISPR-associated) genes form the CRISPR–Cas immune system, which provides sequence-specific adaptive immunity against foreign genetic elements in bacteria and archaea. Immunity is acquired by the integration of short stretches of invasive DNA as novel ‘spacers’ into CRISPR loci. Subsequently, these immune markers are transcribed and generate small non-coding interfering RNAs that specifically guide nucleases for sequence-specific cleavage of complementary sequences. Among the four CRISPR–Cas systems present in Streptococcus thermophilus, CRISPR1 and CRISPR3 have the ability to readily acquire new spacers following bacteriophage or plasmid exposure. In order to investigate the impact of building CRISPR-encoded immunity on the host chromosome, we determined the genome sequence of a BIM (bacteriophage-insensitive mutant) derived from the DGCC7710 model organism, after four consecutive rounds of bacteriophage challenge. As expected, active CRISPR loci evolved via polarized addition of several novel spacers following exposure to bacteriophages. Although analysis of the draft genome sequence revealed a variety of SNPs (single nucleotide polymorphisms) and INDELs (insertions/deletions), most of the in silico differences were not validated by Sanger re-sequencing. In addition, two SNPs and two small INDELs were identified and tracked in the intermediate variants. Overall, building CRISPR-encoded immunity does not significantly affect the genome, which allows the maintenance of important functional properties in isogenic CRISPR mutants. This is critical for the development and formulation of sustainable and robust next-generation starter cultures with increased industrial lifespans.
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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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Ramachandran, Rajesh. "CRISPR/Cas9 System." Resonance 25, no. 12 (December 2020): 1669–80. http://dx.doi.org/10.1007/s12045-020-1088-6.
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Tong, Yaojun, Christopher M. Whitford, Helene L. Robertsen, Kai Blin, Tue S. Jørgensen, Andreas K. Klitgaard, Tetiana Gren, Xinglin Jiang, Tilmann Weber, and Sang Yup Lee. "Highly efficient DSB-free base editing for streptomycetes with CRISPR-BEST." Proceedings of the National Academy of Sciences 116, no. 41 (September 23, 2019): 20366–75. http://dx.doi.org/10.1073/pnas.1913493116.
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Streptomycetes serve as major producers of various pharmacologically and industrially important natural products. Although CRISPR-Cas9 systems have been developed for more robust genetic manipulations, concerns of genome instability caused by the DNA double-strand breaks (DSBs) and the toxicity of Cas9 remain. To overcome these limitations, here we report development of the DSB-free, single-nucleotide–resolution genome editing system CRISPR-BEST (CRISPR-Base Editing SysTem), which comprises a cytidine (CRISPR-cBEST) and an adenosine (CRISPR-aBEST) deaminase-based base editor. Specifically targeted by an sgRNA, CRISPR-cBEST can efficiently convert a C:G base pair to a T:A base pair and CRISPR-aBEST can convert an A:T base pair to a G:C base pair within a window of approximately 7 and 6 nucleotides, respectively. CRISPR-BEST was validated and successfully used in different Streptomyces species. Particularly in nonmodel actinomycete Streptomyces collinus Tü365, CRISPR-cBEST efficiently inactivated the 2 copies of kirN gene that are in the duplicated kirromycin biosynthetic pathways simultaneously by STOP codon introduction. Generating such a knockout mutant repeatedly failed using the conventional DSB-based CRISPR-Cas9. An unbiased, genome-wide off-target evaluation indicates the high fidelity and applicability of CRISPR-BEST. Furthermore, the system supports multiplexed editing with a single plasmid by providing a Csy4-based sgRNA processing machinery. To simplify the protospacer identification process, we also updated the CRISPy-web (https://crispy.secondarymetabolites.org), and now it allows designing sgRNAs specifically for CRISPR-BEST applications.
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Grüschow, Sabine, Januka S. Athukoralage, Shirley Graham, Tess Hoogeboom, and Malcolm F. White. "Cyclic oligoadenylate signalling mediates Mycobacterium tuberculosis CRISPR defence." Nucleic Acids Research 47, no. 17 (August 8, 2019): 9259–70. http://dx.doi.org/10.1093/nar/gkz676.
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Abstract The CRISPR system provides adaptive immunity against mobile genetic elements (MGE) in prokaryotes. In type III CRISPR systems, an effector complex programmed by CRISPR RNA detects invading RNA, triggering a multi-layered defence that includes target RNA cleavage, licencing of an HD DNA nuclease domain and synthesis of cyclic oligoadenylate (cOA) molecules. cOA activates the Csx1/Csm6 family of effectors, which degrade RNA non-specifically to enhance immunity. Type III systems are found in diverse archaea and bacteria, including the human pathogen Mycobacterium tuberculosis. Here, we report a comprehensive analysis of the in vitro and in vivo activities of the type III-A M. tuberculosis CRISPR system. We demonstrate that immunity against MGE may be achieved predominantly via a cyclic hexa-adenylate (cA6) signalling pathway and the ribonuclease Csm6, rather than through DNA cleavage by the HD domain. Furthermore, we show for the first time that a type III CRISPR system can be reprogrammed by replacing the effector protein, which may be relevant for maintenance of immunity in response to pressure from viral anti-CRISPRs. These observations demonstrate that M. tuberculosis has a fully-functioning CRISPR interference system that generates a range of cyclic and linear oligonucleotides of known and unknown functions, potentiating fundamental and applied studies.
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Yuan, Bowei, Congcong Yuan, Lulu Li, Miao Long, and Zeliang Chen. "Application of the CRISPR/Cas System in Pathogen Detection: A Review." Molecules 27, no. 20 (October 18, 2022): 6999. http://dx.doi.org/10.3390/molecules27206999.
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Early and rapid diagnosis of pathogens is important for the prevention and control of epidemic disease. The polymerase chain reaction (PCR) technique requires expensive instrument control, a special test site, complex solution treatment steps and professional operation, which can limit its application in practice. The pathogen detection method based on the clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated protein (CRISPR/Cas) system is characterized by strong specificity, high sensitivity and convenience for detection, which is more suitable for practical applications. This article first reviews the CRISPR/Cas system, and then introduces the application of the two types of systems represented by Type II (cas9), Type V (cas12a, cas12b, cas14a) and Type VI (cas13a) in pathogen detection. Finally, challenges and prospects are proposed.
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Shen, Yucong. "CRISPR/Cas system: A powerful tool for de-extinction." Theoretical and Natural Science 20, no. 1 (December 20, 2023): 227–31. http://dx.doi.org/10.54254/2753-8818/20/20230774.
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The rapid advances in CRISPR technology have opened up new avenues of research and provided hope for a future where extinct species can be brought back to life. De-extinction, the process of resurrecting extinct species by using genetic engineering techniques, is one potential application of CRISPR technology that has gained increasing attention in recent years. The idea is to recover the genetic information of extinct species from preserved tissue samples and recreate them using modern genetic engineering techniques. George Church, a geneticist at Harvard University, is one of the leading scientists exploring the potential of CRISPR for de-extinction. He has spearheaded efforts to resurrect the woolly mammoth, which went extinct over 4,000 years ago, by inserting mammoth DNA into the genome of the Asian elephant. While this project is still in its early stages, it has sparked renewed interest in using CRISPR to bring back other extinct species, such as the Pyrenean ibex, the passenger pigeon, and the Tasmanian tiger. This review paper aims to explore the potential of CRISP technology for de-extinction, including its technical and ethical challenges, and the progress that has been made in various de-extinction projects involving CRISPR technology.
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Eitzinger, Simon, Amina Asif, Kyle E. Watters, Anthony T. Iavarone, Gavin J. Knott, Jennifer A. Doudna, and Fayyaz ul Amir Afsar Minhas. "Machine learning predicts new anti-CRISPR proteins." Nucleic Acids Research 48, no. 9 (April 14, 2020): 4698–708. http://dx.doi.org/10.1093/nar/gkaa219.
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Abstract The increasing use of CRISPR–Cas9 in medicine, agriculture, and synthetic biology has accelerated the drive to discover new CRISPR–Cas inhibitors as potential mechanisms of control for gene editing applications. Many anti-CRISPRs have been found that inhibit the CRISPR–Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method to aid direct identification of new potential anti-CRISPRs using only protein sequence information. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking. We then applied AcRanker to predict candidate anti-CRISPRs from predicted prophage regions within self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA20 (ML1) and AcrIIA21 (ML8). We show that AcrIIA20 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA21 inhibits SpyCas9, Streptococcus aureus Cas9 (SauCas9) and SinCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.
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Zhang, Jing, and Malcolm F. White. "Hot and crispy: CRISPR–Cas systems in the hyperthermophile Sulfolobus solfataricus." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1422–26. http://dx.doi.org/10.1042/bst20130031.
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The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR–Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus.
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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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Степаненко, Liliya Stepanenko, Парамонов, Aleksey Paramonov, Колбасеева, Olga Kolbaseeva, Воскресенская, et al. "BIoInfoRmatIonal analySIS of YersiniapseudotuberculosisIP32953 CRISPR/CaSSyStem." Бюллетень Восточно-Сибирского научного центра Сибирского отделения Российской академии медицинских наук 1, no. 5 (December 6, 2016): 64–67. http://dx.doi.org/10.12737/23384.
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The results of this study include Yersinia pseudotuberculosis CRISPR/Cas system structure analysis. CRISPR/Cas system is a specific adaptive protection against heterogeneous genetic elements. The object of research was the complete genome of Y. pseudotuberculosis IP32953 (NC_006155). CRISPR/Cas system screening was performed by program modelling methods MacSyFinder ver. 1.0.2. CRISPR loci screening and analyzing were carried out by program package: CRISPR Recognition tool (CRT), CRISPI: a CRISPR Interactive database, CRISPRFinder, and PilerCR. Spacer sequences were used in order to find protospacers in ACLAME, GenBank-Phage and RefSeq-Plasmid databases by BLASTn search algorithm. Protospacer sequences could be found in genomes of phages, plasmids and bacteria. In last case complete genomes of bacteria were analyzed by online-tool PHAST: PHAge Search Tool. Y. pseudotuberculosis IP329353 has CRISPR/Cas system that consists of one sequence of cas-genes and three loci. These loci are far away from each other. Locus YP1 is situated in close proximity to cas-genes. Protospacers were found in genomes of Y. pseudotuberculosis PB1/+, Y. intermedia Y228, Y. similis str. 228, Salmonella phage, Enterobacteria phage, Y. pseudotuberculosis IP32953 plasmid pYV and plasmid of Y. pseudotuberculosis IP31758. Thus, the combination of four program methods allows finding CRISPR/Cas system more precisely. Spacer sequences could be used for protospacer screening.
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Sunusi, M., Lurwanu, Y., Halidu, J., and Musa, H. "Crispr Cas System in Plant Genome Editing a New Opportunity in Agriculture to Boost Crop Yield." UMYU Journal of Microbiology Research (UJMR) 3, no. 1 (June 30, 2018): 104–14. http://dx.doi.org/10.47430/ujmr.1831.017.
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Clustered regularly interspaced short palindromic repeats CRISPR/Cas9 technology evolved from a type II bacterial immune system develop in 2013 This system employs RNA-guided nuclease, CRISPR associated (Cas9) to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. The system has the ability to detect specific sequences of letters within the genetic code and to cut DNA at a specific point. Simultaneously with other sequence-specific nucleases, CRISPR/ Cas9 have already breach the boundaries and made genetic engineering much more versatile, efficient and easy also it has been reported to have increased rice grain yield up to 25-30 %, and increased tomato fruits size, branching architecture, and overall plant shape. CRISPR/ Cas also mediated virus resistance in many agricultural crops. In this article, we reviewed the history of the CRISPR/Cas9 system invention and its genome-editing mechanism. We also described the most recent innovation of the CRISPR/Cas9 technology, particularly the broad applications of modified Cas9 variants, and discuss the potential of this system for targeted genome editing and modification for crop improvement.
Abbreviations: CRISPR, clustered regularly interspaced short palindromic repeats; Cas, CRISPR associated; crRNA, CRISPR RNA; tracrRNA, trans-activating crRNA; PAM, protospacer adjacent motif; sgRNA, single guide RNA; gRNA, guide RNA; ssODN, single-stranded DNA oligonucleotide; DSB, double-strand break; NHEJ, non-homologous end joining; HDR, homology directed repair, CRISPRi ,CRISPR interference
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Mohammadi Ghanbarlou, Mahdi, Shahriyar Abdoli, Hamed Omid, Leila Qazizadeh, Hadi Bamehr, Mozhgan Raigani, Hosein Shahsavarani, Morteza Karimipour, and Mohammad Ali Shokrgozar. "Delivery of dCas9 Activator System Using Magnetic Nanoparticles Technology as a Vector Delivery Method for Human Skin Fibroblast." Magnetochemistry 9, no. 3 (February 28, 2023): 71. http://dx.doi.org/10.3390/magnetochemistry9030071.
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The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.
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Selle, Kurt, Todd R. Klaenhammer, and Rodolphe Barrangou. "CRISPR-based screening of genomic island excision events in bacteria." Proceedings of the National Academy of Sciences 112, no. 26 (June 15, 2015): 8076–81. http://dx.doi.org/10.1073/pnas.1508525112.
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Genomic analysis ofStreptococcus thermophilusrevealed that mobile genetic elements (MGEs) likely contributed to gene acquisition and loss during evolutionary adaptation to milk. Clustered regularly interspaced short palindromic repeats–CRISPR-associated genes (CRISPR-Cas), the adaptive immune system in bacteria, limits genetic diversity by targeting MGEs including bacteriophages, transposons, and plasmids. CRISPR-Cas systems are widespread in streptococci, suggesting that the interplay between CRISPR-Cas systems and MGEs is one of the driving forces governing genome homeostasis in this genus. To investigate the genetic outcomes resulting from CRISPR-Cas targeting of integrated MGEs,in silicoprediction revealed four genomic islands without essential genes in lengths from 8 to 102 kbp, totaling 7% of the genome. In this study, the endogenous CRISPR3 type II system was programmed to target the four islands independently through plasmid-based expression of engineered CRISPR arrays. TargetinglacZwithin the largest 102-kbp genomic island was lethal to wild-type cells and resulted in a reduction of up to 2.5-log in the surviving population. Genotyping of Lac−survivors revealed variable deletion events between the flanking insertion-sequence elements, all resulting in elimination of the Lac-encoding island. Chimeric insertion sequence footprints were observed at the deletion junctions after targeting all of the four genomic islands, suggesting a common mechanism of deletion via recombination between flanking insertion sequences. These results established that self-targeting CRISPR-Cas systems may direct significant evolution of bacterial genomes on a population level, influencing genome homeostasis and remodeling.
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Jwair, Noor A., Mushtak T. S. Al-Ouqaili, and Farah Al-Marzooq. "Inverse Association between the Existence of CRISPR/Cas Systems with Antibiotic Resistance, Extended Spectrum β-Lactamase and Carbapenemase Production in Multidrug, Extensive Drug and Pandrug-Resistant Klebsiella pneumoniae." Antibiotics 12, no. 6 (May 29, 2023): 980. http://dx.doi.org/10.3390/antibiotics12060980.
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Antimicrobial resistance, with the production of extended-spectrum β-lactamases (ESBL) and carbapenemases, is common in the opportunistic pathogen, Klebsiella pneumoniae. This organism has a genome that can contain clustered regularly interspaced short palindromic repeats (CRISPRs), which operate as a defense mechanism against external invaders such as plasmids and viruses. This study aims to determine the association of the CRISPR/Cas systems with antibiotic resistance in K. pneumoniae isolates from Iraqi patients. A total of 100 K. pneumoniae isolates were collected and characterized according to their susceptibility to different antimicrobial agents. The CRISPR/Cas systems were detected via PCR. The phenotypic detection of ESBLs and carbapenemases was performed. The production of ESBL was detected in 71% of the isolates. Carbapenem-resistance was detected in 15% of the isolates, while only 14% were susceptible to all antimicrobial agents. Furthermore, the bacteria were classified into multidrug (77%), extensively drug-resistant (11.0%) and pandrug-resistant (4.0%). There was an inverse association between the presence of the CRISPR/Cas systems and antibiotic resistance, as resistance was higher in the absence of the CRISPR/Cas system. Multidrug resistance in ESBL-producing and carbapenem-resistant K. pneumoniae occurred more frequently in strains negative for the CRISPR/Cas system. Thus, we conclude that genes for exogenous antibiotic resistance can be acquired in the absence of the CRISPR/Cas modules that can protect the bacteria against acquiring foreign DNA.
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Wang, Jiawei, Wei Dai, Jiahui Li, Ruopeng Xie, Rhys A. Dunstan, Christopher Stubenrauch, Yanju Zhang, and Trevor Lithgow. "PaCRISPR: a server for predicting and visualizing anti-CRISPR proteins." Nucleic Acids Research 48, W1 (May 27, 2020): W348—W357. http://dx.doi.org/10.1093/nar/gkaa432.
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Abstract Anti-CRISPRs are widespread amongst bacteriophage and promote bacteriophage infection by inactivating the bacterial host's CRISPR–Cas defence system. Identifying and characterizing anti-CRISPR proteins opens an avenue to explore and control CRISPR–Cas machineries for the development of new CRISPR–Cas based biotechnological and therapeutic tools. Past studies have identified anti-CRISPRs in several model phage genomes, but a challenge exists to comprehensively screen for anti-CRISPRs accurately and efficiently from genome and metagenome sequence data. Here, we have developed an ensemble learning based predictor, PaCRISPR, to accurately identify anti-CRISPRs from protein datasets derived from genome and metagenome sequencing projects. PaCRISPR employs different types of feature recognition united within an ensemble framework. Extensive cross-validation and independent tests show that PaCRISPR achieves a significantly more accurate performance compared with homology-based baseline predictors and an existing toolkit. The performance of PaCRISPR was further validated in discovering anti-CRISPRs that were not part of the training for PaCRISPR, but which were recently demonstrated to function as anti-CRISPRs for phage infections. Data visualization on anti-CRISPR relationships, highlighting sequence similarity and phylogenetic considerations, is part of the output from the PaCRISPR toolkit, which is freely available at http://pacrispr.erc.monash.edu/.
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Akhmetova, E. A., V. M. Golyshev, I. P. Vokhtantcev, M. I. Meschaninova, A. G. Venyaminova, and D. S. Novopashina. "Photoactivatable CRISPR/Cas9 System." Russian Journal of Bioorganic Chemistry 47, no. 2 (March 2021): 496–504. http://dx.doi.org/10.1134/s1068162021020023.
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Schindele, Patrick, Felix Wolter, and Holger Puchta. "Das CRISPR/Cas-System." Biologie in unserer Zeit 48, no. 2 (April 2018): 100–105. http://dx.doi.org/10.1002/biuz.201810642.
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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.
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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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Gong, Chongzhi, Shengchan Huang, Rentao Song, and Weiwei Qi. "Comparative Study between the CRISPR/Cpf1 (Cas12a) and CRISPR/Cas9 Systems for Multiplex Gene Editing in Maize." Agriculture 11, no. 5 (May 10, 2021): 429. http://dx.doi.org/10.3390/agriculture11050429.
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Although the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been proved to be an efficient multiplex gene editing system in maize, it was still unclear how CRISPR/Cpf1 (Cas12a) system would perform for multiplex gene editing in maize. To this end, this study compared the CRISPR/Cpf1 system and CRISPR/Cas9 system for multiplex gene editing in maize. The bZIP transcription factor Opaque2 (O2) was used as the target gene in both systems. We found that in the T0 and T1 generations, the CRISPR/Cpf1 system showed lower editing efficiency than the CRISPR/Cas9 system. However, in the T2 generation, the CRISPR/Cpf1 system generated more types of new mutations. While the CRISPR/Cas9 system tended to edit within the on-target range, the CRISPR/Cpf1 system preferred to edit in between the targets. We also found that in the CRISPR/Cpf1 system, the editing efficiency positively correlated with the expression level of Cpf1. In conclusion, the CRISPR/Cpf1 system offers alternative choices for target-site selection for multiplex gene editing and has acceptable editing efficiency in maize and is a valuable alternative choice for gene editing in crops.
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Maier, Lisa-Katharina, Britta Stoll, Jutta Brendel, Susan Fischer, Friedhelm Pfeiffer, Mike Dyall-Smith, and Anita Marchfelder. "The ring of confidence: a haloarchaeal CRISPR/Cas system." Biochemical Society Transactions 41, no. 1 (January 29, 2013): 374–78. http://dx.doi.org/10.1042/bst20120263.
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To survive the constant invasions by foreign genetic elements, prokaryotes have evolved various defensive systems. Almost all sequenced archaea, and half of the analysed bacteria use the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system, a recently identified prokaryotic immune system that can fend off invading elements in a sequence-specific manner. Few archaeal CRISPR/Cas systems have been analysed so far, and the molecular details of many of the steps involved in adaptation and defence are yet to be understood. In the present paper, we summarize our current knowledge about the CRISPR/Cas system in Haloferax volcanii, an extremely halophilic archaeon that was isolated from the Dead Sea. H. volcanii encodes a type I-B CRISPR/Cas system, and carries three CRISPR loci and eight Cas proteins. Although in laboratory culture for more than three decades, this defence system was shown to be still active. All three CRISPR loci are transcribed and processed into mature crRNAs (CRISPR RNAs). Cells challenged with engineered plasmids can recognize and eliminate these invading elements if they contain the correct PAM (protospacer adjacent motif) and a sequence that can be recognized by one of the CRISPR spacers.
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Osakabe, Keishi, Naoki Wada, Emi Murakami, Naoyuki Miyashita, and Yuriko Osakabe. "Genome editing in mammalian cells using the CRISPR type I-D nuclease." Nucleic Acids Research 49, no. 11 (June 2, 2021): 6347–63. http://dx.doi.org/10.1093/nar/gkab348.
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Abstract Adoption of CRISPR–Cas systems, such as CRISPR–Cas9 and CRISPR–Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I—the most abundant CRISPR system in bacteria—remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense (‘Cascade’: Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR–Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR–Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR–Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.
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Westbrook, Adam W., Murray Moo-Young, and C. Perry Chou. "Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis." Applied and Environmental Microbiology 82, no. 16 (June 3, 2016): 4876–95. http://dx.doi.org/10.1128/aem.01159-16.
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ABSTRACTThe establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction inBacillus subtilisis essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering inB. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed forEscherichia coliandSaccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery.IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system inBacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering ofB. subtilisstrains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.
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Jing, Yike. "Applications and Prospects of CRISPR-Cas system in Cyanobacteria." BIO Web of Conferences 61 (2023): 01009. http://dx.doi.org/10.1051/bioconf/20236101009.
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Cyanobacteria are prokaryotic microorganisms with capacity to perform photosynthesis and provide valuable platform to produce high-value bioactive compounds in carbon-neutral pathway. However, due to the relative lack of high throughput genetic manipulation tools for cyanobacteria, their usage for complexity chemicals is lagging behind. The gene editing technology based on the CRISPR-Cas system has the advantage of simplicity and efficiency in recent years, making it a new tool for synthetic biology of cyanobacteria. In this review, we first introduced the CRISPR-Cas system and the types of CRISPR-Cas in cyanobacteria. In addition, we review the development of CRISPR-based systems in cyanobacteria, including the use of CRISPR systems for gene integration, deletion, and transcriptional regulation. Meanwhile, the CRISPR system can couple different functional proteins to achieve point mutations, such as base editing. Finally, we discuss the current limitations of CRISPR technology and its future application prospects in cyanobacteria to make an outlook. Finally, we discussed the current limitations of CRISPR technology and its future application prospects in cyanobacteria.
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Aslam, Shakira, Ali Umair, Zaid Aslam, Muhammad Zafar Saleem, and Hamid Bashir. "CRISPR/Cas System: An Effective Tool Against Pathogenic Diseases." Postępy Mikrobiologii - Advancements of Microbiology 62, no. 2 (June 1, 2023): 87–99. http://dx.doi.org/10.2478/am-2023-0009.
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Abstract Viral pathogens are major concern nowadays. Bacterial CRISPR/Cas systems help in defending the host body against different pathogens including viruses too. This system contains restriction enzymes that introduce dsDNA breaks on target site to make the virus non-functional by damaging its genes. Coronavirus, HIV and Herpes viruses are causing mortality all around the world. To control the spread of disease, early detection and treatment is required. CRISPR system due to its high efficiency provides a platform to restrict the viral entry into host cell, viral genome editing and eliminate the latent infections. In this review, the CRISPR strategies against three major viral diseases are put under consideration: Coronavirus, Acquired Immunodeficiency Syndrome and Herpesvirus associated diseases. Three CRISPR/Cas systems have been discussed including CRISPR/Cas12, CRISPR/Cas9 and CRISPR/Cas13 that are used against the above listed viruses.
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Haider, Muhammad Zulqarnain, Muhammad Abu Bakr Shabbir, Tahir Yaqub, Adeel Sattar, Muhammad Kashif Maan, Sammina Mahmood, Tahir Mehmood, and Hassaan Bin Aslam. "CRISPR-Cas System: An Adaptive Immune System’s Association with Antibiotic Resistance in Salmonella enterica Serovar Enteritidis." BioMed Research International 2022 (March 28, 2022): 1–7. http://dx.doi.org/10.1155/2022/9080396.
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Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- (CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.
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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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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.
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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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McInally, S. G., K. D. Hagen, C. Nosala, J. Williams, K. Nguyen, J. Booker, K. Jones, and Scott C. Dawson. "Robust and stable transcriptional repression in Giardia using CRISPRi." Molecular Biology of the Cell 30, no. 1 (January 2019): 119–30. http://dx.doi.org/10.1091/mbc.e18-09-0605.
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Giardia lamblia is a binucleate protistan parasite causing significant diarrheal disease worldwide. An inability to target Cas9 to both nuclei, combined with the lack of nonhomologous end joining and markers for positive selection, has stalled the adaptation of CRISPR/Cas9-mediated genetic tools for this widespread parasite. CRISPR interference (CRISPRi) is a modification of the CRISPR/Cas9 system that directs catalytically inactive Cas9 (dCas9) to target loci for stable transcriptional repression. Using a Giardia nuclear localization signal to target dCas9 to both nuclei, we developed efficient and stable CRISPRi-mediated transcriptional repression of exogenous and endogenous genes in Giardia. Specifically, CRISPRi knockdown of kinesin-2a and kinesin-13 causes severe flagellar length defects that mirror defects with morpholino knockdown. Knockdown of the ventral disk MBP protein also causes severe structural defects that are highly prevalent and persist in the population more than 5 d longer than defects associated with transient morpholino-based knockdown. By expressing two guide RNAs in tandem to simultaneously knock down kinesin-13 and MBP, we created a stable dual knockdown strain with both flagellar length and disk defects. The efficiency and simplicity of CRISPRi in polyploid Giardia allows rapid evaluation of knockdown phenotypes and highlights the utility of CRISPRi for emerging model systems.
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He, Yuxuan, Wei Yan, Likun Long, Liming Dong, Yue Ma, Congcong Li, Yanbo Xie, et al. "The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection." Genes 14, no. 4 (March 31, 2023): 850. http://dx.doi.org/10.3390/genes14040850.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) are promising molecular diagnostic tools for rapidly and precisely elucidating the structure and function of genomes due to their high specificity, programmability, and multi-system compatibility in nucleic acid recognition. Multiple parameters limit the ability of a CRISPR/Cas system to detect DNA or RNA. Consequently, it must be used in conjunction with other nucleic acid amplification techniques or signal detection techniques, and the reaction components and reaction conditions should be modified and optimized to maximize the detection performance of the CRISPR/Cas system against various targets. As the field continues to develop, CRISPR/Cas systems have the potential to become an ultra-sensitive, convenient, and accurate biosensing platform for the detection of specific target sequences. The design of a molecular detection platform employing the CRISPR/Cas system is asserted on three primary strategies: (1) Performance optimization of the CRISPR/Cas system; (2) enhancement of the detection signal and its interpretation; and (3) compatibility with multiple reaction systems. This article focuses on the molecular characteristics and application value of the CRISPR/Cas system and reviews recent research progress and development direction from the perspectives of principle, performance, and method development challenges to provide a theoretical foundation for the development and application of the CRISPR/CAS system in molecular detection technology.
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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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Sorokin, Valery A., Mikhail S. Gelfand, and Irena I. Artamonova. "Evolutionary Dynamics of Clustered Irregularly Interspaced Short Palindromic Repeat Systems in the Ocean Metagenome." Applied and Environmental Microbiology 76, no. 7 (January 29, 2010): 2136–44. http://dx.doi.org/10.1128/aem.01985-09.
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ABSTRACT Clustered regularly interspaced short palindromic repeats (CRISPRs) form a recently characterized type of prokaryotic antiphage defense system. The phage-host interactions involving CRISPRs have been studied in experiments with selected bacterial or archaeal species and, computationally, in completely sequenced genomes. However, these studies do not allow one to take prokaryotic population diversity and phage-host interaction dynamics into account. This gap can be filled by using metagenomic data: in particular, the largest existing data set, generated from the Sorcerer II Global Ocean Sampling expedition. The application of three publicly available CRISPR recognition programs to the Global Ocean metagenome produced a large proportion of false-positive results. To address this problem, a filtering procedure was designed. It resulted in about 200 reliable CRISPR cassettes, which were then studied in detail. The repeat consensuses were clustered into several stable classes that differed from the existing classification. Short fragments of DNA similar to the cassette spacers were more frequently present in the same geographical location than in other locations (P, <0.0001). We developed a catalogue of elementary CRISPR-forming events and reconstructed the likely evolutionary history of cassettes that had common spacers. Metagenomic collections allow for relatively unbiased analysis of phage-host interactions and CRISPR evolution. The results of this study demonstrate that CRISPR cassettes retain the memory of the local virus population at a particular ocean location. CRISPR evolution may be described using a limited vocabulary of elementary events that have a natural biological interpretation.
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Pavlova, Yekaterina S., David Paez-Espino, Andrew Yu Morozov, and Ilya S. Belalov. "Searching for fat tails in CRISPR-Cas systems: Data analysis and mathematical modeling." PLOS Computational Biology 17, no. 3 (March 26, 2021): e1008841. http://dx.doi.org/10.1371/journal.pcbi.1008841.
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Understanding CRISPR-Cas systems—the adaptive defence mechanism that about half of bacterial species and most of archaea use to neutralise viral attacks—is important for explaining the biodiversity observed in the microbial world as well as for editing animal and plant genomes effectively. The CRISPR-Cas system learns from previous viral infections and integrates small pieces from phage genomes called spacers into the microbial genome. The resulting library of spacers collected in CRISPR arrays is then compared with the DNA of potential invaders. One of the most intriguing and least well understood questions about CRISPR-Cas systems is the distribution of spacers across the microbial population. Here, using empirical data, we show that the global distribution of spacer numbers in CRISPR arrays across multiple biomes worldwide typically exhibits scale-invariant power law behaviour, and the standard deviation is greater than the sample mean. We develop a mathematical model of spacer loss and acquisition dynamics which fits observed data from almost four thousand metagenomes well. In analogy to the classical ‘rich-get-richer’ mechanism of power law emergence, the rate of spacer acquisition is proportional to the CRISPR array size, which allows a small proportion of CRISPRs within the population to possess a significant number of spacers. Our study provides an alternative explanation for the rarity of all-resistant super microbes in nature and why proliferation of phages can be highly successful despite the effectiveness of CRISPR-Cas systems.
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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.
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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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Mimoune, Nora, Oumayma Benadjel, Ratiba Baazizi, and Djamel Kelef. "CRISPR/Cas9 uses." Veterinarska stanica 52, no. 4 (February 22, 2021): 369–86. http://dx.doi.org/10.46419/vs.52.4.9.
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Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR- associated system (Cas) is a system that provides immunity to most prokaryoticorganisms against viral attacks and other foreign bodies. CRISPR systems consist of a scissor-like protein called Cas9 and a genetic GPS guide “The guide RNA”. However, researchers have reoriented and repurposed the primordial immune system to precisely manipulate genomes in most organisms by introducing DNA double-strand breaks at specific genome locations to introduce specific DNA modifications. More applications of CRISPR have arisen since its discovery, from disabling parasites to correcting mutations and improving crop yields. This review was conceived as a guide to CRISPR technology, from its discovery to the latest breakthroughs. It is hoped that this study will provide a general-based view for this life changing technology, inspiring scientists to go further with CRISPR in the sake of a better life.
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Hegde, Shivanand, Hallie E. Rauch, Grant L. Hughes, and Nikki Shariat. "Identification and characterization of two CRISPR/Cas systems associated with the mosquito microbiome." Access Microbiology 5, no. 8 (August 1, 2023). http://dx.doi.org/10.1099/acmi.0.000599.v4.
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The microbiome profoundly influences many traits in medically relevant vectors such as mosquitoes, and a greater functional understanding of host–microbe interactions may be exploited for novel microbial-based approaches to control mosquito-borne disease. Here, we characterized two novel clustered regularly interspaced short palindromic repeats (CRISPR)/Cas systems in Serratia sp. Ag1, which was isolated from the gut of an Anopheles gambiae mosquito. Two distinct CRISPR/Cas systems were identified in Serratia Ag1, CRISPR1 and CRISPR2. Based on cas gene composition, CRISPR1 is classified as a type I-E CRISPR/Cas system and has a single array, CRISPR1. CRISPR2 is a type I-F system with two arrays, CRISPR2.1 and CRISPR2.2. RT-PCR analyses show that all cas genes from both systems are expressed during logarithmic growth in culture media. The direct repeat sequences of CRISPRs 2.1 and 2.2 are identical and found in the arrays of other Serratia spp., including S. marcescens and S. fonticola , whereas CRISPR1 is not. We searched for potential spacer targets and revealed an interesting difference between the two systems: only 9 % of CRISPR1 (type I-E) targets are in phage sequences and 91 % are in plasmid sequences. Conversely, ~66 % of CRISPR2 (type I-F) targets are found within phage genomes. Our results highlight the presence of CRISPR loci in gut-associated bacteria of mosquitoes and indicate interplay between symbionts and invasive mobile genetic elements over evolutionary time.
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Ma, Shuai, Feiyu Wang, Zhang Xuejing, Qiao Liping, Guo Xueping, Xuemei Lu, and Qingsheng Qi. "Repurposing endogenous type II CRISPR‐Cas9 system for genome editing in Streptococcus thermophilus." Biotechnology and Bioengineering, November 23, 2023. http://dx.doi.org/10.1002/bit.28608.
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AbstractStreptococcus thermophilus has been extensively used in industrial milk fermentation. However, lack of efficient genetic manipulation approaches greatly hampered the industrial application of this species. Here, we repurposed the endogenous CRISPR1 and CRISPR3 systems, both belong to type II‐A CRISPR‐Cas9, by delivering a self‐targeting CRISPR array with DNA repair template into S. thermophilus LMD‐9. We achieved 785‐bp deletion in lacZ gene by repurposing CRISPR1 and CRISPR3 systems with efficiencies of 35% and 59%, respectively, when 1‐kb DNA repair template was provided. While providing with 1.5‐kb repair template, the editing efficiency for deletion in lacZ gene reached 90% using CRISPR3 systems. Diverse editing outcomes encompassing a stop code insertion and single nucleotide variation within lacZ, as well as a 234‐bp DNA fragment insertion upstream of ster_0903, were generated with high efficiencies of 75%–100% using the CRISPR3 system. Harnessing the customized endogenous CRISPR3 system to target six genes of eps gene cluster, we obtained six single‐gene knockout mutants with efficiencies of 29%–80%, and proved that the epsA, epsE, and epsG were the key genes affecting exopolysaccharides biosynthesis in S. thermophilus LMD‐9. Altogether, repurposing the native type II‐A CRISPR‐Cas9 can be served as a toolkit for precise genome engineering in S. thermophilus for biotechnological applications.
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Feng, Qing, Xiaoyu Ning, Lei Qin, Jun Li, and Chun Li. "Quantitative and modularized CRISPR/dCas9-dCpf1 dual function system in Saccharomyces cerevisiae." Frontiers in Bioengineering and Biotechnology 11 (October 18, 2023). http://dx.doi.org/10.3389/fbioe.2023.1218832.
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Introduction: Both CRISPR/dCas9 and CRISPR/dCpf1 genome editing systems have shown exciting promises in modulating yeast cell metabolic pathways. However, each system has its deficiencies to overcome. In this study, to achieve a compensatory effect, we successfully constructed a dual functional CRISPR activation/inhibition (CRISPRa/i) system based on Sp-dCas9 and Fn-dCpf1 proteins, along with their corresponding complementary RNAs.Methods: We validated the high orthogonality and precise quantity targeting of selected yeast promoters. Various activating effector proteins (VP64, p65, Rta, and VP64-p65-Rta) and inhibiting effector proteins (KRAB, MeCP2, and KRAB-MeCP2), along with RNA scaffolds of MS2, PP7 and crRNA arrays were implemented in different combinations to investigate quantitative promoter strength. In the CRISPR/dCas9 system, the regulation rate ranged from 81.9% suppression to 627% activation in the mCherry gene reporter system. Studies on crRNA point mutations and crRNA arrays were conducted in the CRISPR/dCpf1 system, with the highest transcriptional inhibitory rate reaching up to 530% higher than the control. Furthermore, the orthogonal CRISPR/dCas9-dCpf1 inhibition system displayed distinct dual functions, simultaneously regulating the mCherry gene by dCas9/gRNA (54.6% efficiency) and eGFP gene by dCpf1/crRNA (62.4% efficiency) without signal crosstalk.Results and discussion: Finally, we established an engineered yeast cell factory for β-carotene production using the CRISPR/dCas9-dCpf1 bifunctional system to achieve targeted modulation of both heterologous and endogenous metabolic pathways in Saccharomyces cerevisiae. The system includes an activation module of CRISPRa/dCas9 corresponding to a gRNA-protein complex library of 136 plasmids, and an inhibition module of CRISPRi/dCpf1 corresponding to a small crRNA array library. Results show that this CRISPR/dCas9-dCpf1 bifunctional orthogonal system is more quantitatively effective and expandable for simultaneous CRISPRa/i network control compared to single-guide edition, demonstrating higher potential of future application in yeast biotechnology.
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Lipatova, Indrė. "Recent doctoral theses (biochemistry, biology, biophysics, ecology and environmental) in Lithuania." Biologija 68, no. 1 (May 4, 2022). http://dx.doi.org/10.6001/biologija.v68i1.4704.
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The research objective of this work was to investigate and study details of the CRISPR adaptation process in several different CRISPR-Cas systems as well as use genome-wide CRISPR screening to elucidate cell-intermedilysin interactions. The results of this study showed that Cas1-Cas2 complex from S. thermophilus CRISPR4-Cas system forms a complex that integrates prespacers into the CRISPR array. DnaQ domain fused to Cas2 in this system is a 3’–5’ DNA exonuclease. DnaQ domain is dispensable for spacer integration; however, it serves to trim back overextended 3’ overhangs of the prespacer. Cas1, Cas2, and Csn2 proteins from S. thermophilus CRISPR3-Cas system form at least three different complexes, which interact with Cas9 from the same system via the DNA tether. The identified complexes from CRISPR3-Cas system represent a spacer capture step of the new spacer acquisition process, as they harbour spacer length DNA in their assemblies. Genome-wide CRISPR screening can reveal novel fundamental biological pathways in the membrane composition and lipid metabolism when used in concert with membrane targeting toxins. Intermedilysin has many more dependency factors than previously known CD59 and cholesterol. Among them are heparan sulfates, glucosylceramides, and many other protein or lipid glycosylation factors. ILY can be inhibited by heparin or the removal of heparan sulfates from cells using bacterial heparinases.