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1
Chaturvedi, Sarika, and Jinny Tomar. "CRISPR/CAS 9 Mediated Treatment for UTIs." International Journal for Modern Trends in Science and Technology 6, no. 5 (2020): 82–94. http://dx.doi.org/10.46501/ijmtst060515.
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“CRISPR" is short and used for "CRISPR-Cas9. CRISPR stands for clustered regularly interspaced short palindromic repeats. CRISPRs are specialized stretches of DNA. The protein Cas9 (or "CRISPR-associated") is an enzyme that acts like a pair of molecular scissors, capable of cutting strands of DNA and can be used in conjunction with engineered CRISPR sequences to hunt down codes and slice into them like a molecular scalpel, allowing geneticists to cut out a target gene, either to remove it or replace it with a new sequence. Therefore it is a simple and powerful tool for editing genomes to easily alter DNA sequences and amend gene function. In 1987, The CRISPR locus was first identified in Escherichia coli and discovered when a genetic structure containing 5 highly homologous repeats of 29 nucleotides separated by 32-nucleotide spacers (Ishino Y 1987).
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Ding, Anqi, Zhongjin Gu, and Zihan Chen. "Application of CRISPR-Cas 9 system in cancer therapy." Highlights in Science, Engineering and Technology 74 (December 29, 2023): 302–6. http://dx.doi.org/10.54097/s50r4154.
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There are several gene editing technologies under the background of that time, such as ZFN and TALEN which are hard to use and have high cost. On the contrary, the efficiency and accuracy of CRISPR-Cas system has attracted the attention of scientists. CRISPR is a Short palindromic repeat sequence, and it is widespread in many prokaryotes. The first CRISPR was cloned in E. coli by scientists in 1987, and several other sequences were subsequently cloned. The Cas is a nuclease, then two of them cooperate to become the CRISPR-Cas system. It is not only an acquired immune defense mechanism of prokaryotes against virus, but also a tool for gene editing. As an emerging gene editing technology in recent years, CRISPR-Cas9 technology has been widely applied to the treatment of a variety of diseases. This review focused on the application of this technology in cancer. This review systematically introduced the working principle and immune mechanism of CRISPR-Cas9 and compared CRISPR-Cas9 technology with two previous generations of gene-editing technology, current application of CRISPR-Cas9 in cancer treatment, elaborating it with relevant examples, and finally pointed out the challenges of CRISPR-Cas9 in cancer treatment, looking forward to the development of this technology in the future. The purpose of the paper is to introduce and popularize CRISPR-Cas9 technology, and to provide a direction for future cancer treatment.
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Mali, Franc. "Is the Patent System the Way Forward with the CRISPR-Cas 9 Technology?" Science & Technology Studies 33, no. 4 (2020): 2–23. http://dx.doi.org/10.23987/sts.70114.
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CRISPR-Cas9 technology is reshaping the way scientists conduct research in genetic engineering. It is predicted to revolutionise not only the fields of medicine, biology, agriculture and industry but, much like all revolutionary technologies of the past, the way humans live. Given the anticipated and already seen benefits of CRISPR-Cas 9 in different areas of human life, this new technology may be defined as a true breakthrough scientific discovery. The article presents several challenges connected with various dimensions of the CRISPR-Cas 9 patent landscape. The central argument is that today the biggest challenge is finding a intermediary way that ensures a balance between providing sufficient openness for the further progress of basic research in CRISPR-Cas 9 such as ‘niche’ areas of the latest genetic engineering and adequate intellectual property rights to incentivise its commercialisation and application. The article contends the endeavours by academic scientific institutions to arrive at short-term benefits of the new CRISPR-Cas 9 technology do not constitute such an intermediary way, especially when the CRISPR-Cas 9 patent landscape is viewed as part of a series of controversial bioethical discussions that have been underway for over 40 years.
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Borisenko, A. Yu, N. A. Arefieva, Yu P. Dzhioev, 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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Jay, Chourasia. "The Engineered CRISPR-CAS System is a Beneficial Biological Tool for Detecting and Combating Antibiotic Resistance Microbes." BOHR International Journal of Biocomputing and Nano Technology 1, no. 1 (2021): 40–41. http://dx.doi.org/10.54646/bijbnt.08.
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Nowadays, the quick detection of antibiotic resistance bacteria causes a major problem in the field of the development of new antibiotics against the resistant bacteria. To overcome this problem, genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) can be used. The CRISPR-CAS system is useful for targeting and killing antibiotic-resistant bacteria by cleaving resistance genes. It is also used to detect antibioticresistant bacteria. CRISPR is made up of a single guide RNA and the CAS 9 protein. The single guide RNA is used to guide toward the target sequence, and the CAS 9 protein is an enzyme that cuts DNA and is used in conjunction with the guide RNA. This modified sgRNA contains a complementary sequence to that of the target resistance gene and recognizes the target resistance sequence; therefore, it is cleaved by CAS-9 protein, and the removal of the resistance gene turns bacteria into antibiotic-sensitive ones. One of the delivery systems of CRISPR into bacteria is via bacteriophage.
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Chourasia, Jay, Pourya Gholizadeh, S¸ kran Kse, et al. "The Engineered CRISPR-CAS System is a Beneficial Biological Tool for Detecting and Combating Antibiotic Resistance Microbes." BOHR International Journal of Biocomputing and Nano Technology 1, no. 1 (2022): 40–41. http://dx.doi.org/10.54646/bijbnt.008.
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Nowadays, the quick detection of antibiotic resistance bacteria causes a major problem in the field of the development of new antibiotics against the resistant bacteria. To overcome this problem, genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) can be used. The CRISPR-CAS system is useful for targeting and killing antibiotic-resistant bacteria by cleaving resistance genes. It is also used to detect antibioticresistant bacteria. CRISPR is made up of a single guide RNA and the CAS 9 protein. The single guide RNA is used to guide toward the target sequence, and the CAS 9 protein is an enzyme that cuts DNA and is used in conjunction with the guide RNA. This modified sgRNA contains a complementary sequence to that of the target resistance gene and recognizes the target resistance sequence; therefore, it is cleaved by CAS-9 protein, and the removal of the resistance gene turns bacteria into antibiotic-sensitive ones. One of the delivery systems of CRISPR into bacteria is via bacteriophage.
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Driehuis, Else, and Hans Clevers. "CRISPR/Cas 9 genome editing and its applications in organoids." American Journal of Physiology-Gastrointestinal and Liver Physiology 312, no. 3 (2017): G257—G265. http://dx.doi.org/10.1152/ajpgi.00410.2016.
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Organoids are three-dimensional (3D) structures derived from adult or embryonic stem cells that maintain many structural and functional features of their respective organ. Recently, genome editing based on the bacterial defense mechanism CRISPR/Cas9 has emerged as an easily applicable and reliable laboratory tool. Combining organoids and CRISPR/Cas9 creates exciting new opportunities to study organ development and human disease in vitro. The potential applications of CRISPR in organoids are only beginning to be explored.
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Austin, Publishing Group. "Role of CRISPR Cas-9 in Thyroid Cancer." Austin Journal of Surgery 9, no. 1 (2022): 1282. https://doi.org/10.26420/austinjsurg.2022.1282.
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Abstract The regularly clustered and interspersed short palindromic repeats CRISPR are used for the treatment against many diseases. It is also widely used in the field of medicine. It can speedily screen the entire genome also facilitates the regulation of gene therapy for the certain diseases due to its strong specificity and high efficiency. CRISPR-CAS 9 can be used in the different field of tumor research for changing the genome to explore the mechanism of tumor development and. It is used for the treatment of tumors and knocks out specific genes. <strong>Keywords:</strong> Anaplastic Carcinoma; Alpha9-nAChR DNA; Cancer; CRISPRCAS 9
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Chourasia, Jay. "The engineered CRISPR-CAS system is a beneficial biological tool for detecting and combating antibiotic resistance microbes." BOHR Journal of Biocomputing and Nano Technology 1, no. 1 (2023): 28–29. http://dx.doi.org/10.54646/bjbnt.2023.05.
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Nowadays, the quick detection of antibiotic resistance bacteria causes a major problem in the field of the development of new antibiotics against the resistant bacteria. To overcome this problem, genome editing tools like clustered regularly interspaced short palindromic repeats (CRISPR) can be used. The CRISPR-CAS system is useful for targeting and killing antibiotic-resistant bacteria by cleaving resistance genes. It is also used to detect antibiotic resistant bacteria. CRISPR is made up of a single guide RNA and the CAS 9 protein. The single guide RNA is used to guide toward the target sequence, and the CAS 9 protein is an enzyme that cuts DNA and is used in conjunction with the guide RNA. This modified sgRNA contains a complementary sequence to that of the target resistance gene and recognizes the target resistance sequence; therefore, it is cleaved by CAS-9 protein, and the removal of the resistance gene turns bacteria into antibiotic-sensitive ones. One of the delivery systems of CRISPR into bacteria is via bacteriophage.
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Liu, Zheshi, and Yuxiang Zhang. "CRISPR/Cas9 in the treatment of -thalassemia." Theoretical and Natural Science 27, no. 1 (2023): 266–70. http://dx.doi.org/10.54254/2753-8818/27/20240745.
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Based on clinical evidence, medication resistance poses a significant challenge to the treatment of cancer. It causes the disease to become uncontrollable and raises death rates. Drug resistance arises from a variety of causes, but a change in the inherited makeup of tumor cells is typically the root reason. The ability to modify the genome is growing with the recent discovery of clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas)9 technology, which may be helpful in reducing drug resistance. Owing to its exceptional accuracy and efficiency, the CRISPR/Cas 9 system has been used to investigate the relevant roles of cancer-causing genes, create animal models of tumors, and identify potential therapeutic targets. As a result, it has emerged as the go-to technique for therapeutic gene editing. Utilizing CRISPR/Cas 9 technologies in the treatment of different diseases is growing. Because oncogene regulation differs from normal gene regulation, the CRISPR/Cas 9 system offers efficient methods for oncogene elimination, interference with expression, and modification of activity, all of which can effectively impede the growth of tumors. This article discusses the potential of the CRISPR/Cas9 system to identify resistance targets in drug-resistant breast cancer and reverse resistance gene alterations. Furthermore, the difficulties that prevent this technology from being clinically applicable and emphasize the CRISPR/Cas9 systems are discussed. The CRISPR/Cas9 system will be a crucial component of personalized medicine and is anticipated to have a significant impact on reducing drug resistance in cancer therapy.
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Masood, Ahmad Jalal, Ejaz Ahmed, Rimsha Iftikhar, Laiba Javed, Aimen Sahar, and Muhammad Hossain. "Applications and Challenges of CRISPER/Cas-9 System in Cancer Therapy." Global Pharmaceutical Sciences Review IX, no. I (2024): 10–28. http://dx.doi.org/10.31703/gpsr.2024(ix-i).02.
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This review article is aimed at providing an outline of the present information base available on the applications of CRISPR/Cas9 in cancer therapy and the challenges raised by its implementation. We will first explain the mechanism of action and other key components pertaining to the functioning of CRISPR/Cas9 technology. Further, we will provide case studies that have had some success in pre-clinical and clinical applications of CRISPR/Cas9. However, there are several challenges that prevent the wide application of this very promising approach of CRISPR/Cas9 in cancer therapy. Among them are off-target effects and unintended mutations, difficulties in delivery, targeting specificity, etc. Specificity enhancement, improvement in delivery systems, and solutions against regulatory challenges have been described.
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Salsabila, Pisqiantin Aenan, Nanda Bulkis, Lalu Denendra Praditama, Nur Ramdhani Kanata, Reivirly Khairadaty Maghfirahandini, and Anggit Listyacahyani Sunarwidhi. "Literature Review: CRISPR-Cas 9 Genetic Engineering as Breast Cancer Therapy." Jurnal Biologi Tropis 25, no. 2 (2025): 1894–902. https://doi.org/10.29303/jbt.v25i2.8957.
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Cancer is a disease characterized by uncontrolled abnormal cell growth, originating from cancer stem cells. These cells form a side population with stemness properties similar to normal stem cells, have high tumorigenicity, and contribute to the development of cancer. Among all cases, breast cancer is one of the causes of cancer death in women worldwide, estimated to reach 28% of new cancers. The application of one of the genetic engineering techniques in the form of CRISPR / Cas9 which can be used as an alternative choice in optimizing breast cancer therapy. This literature review aims to determine CRISPR-Cas 9 Genetic Engineering as Breast Cancer Therapy. This journal review method is through searching for articles from databases such as PubMed, ScienceDirect, and Google Scholar using relevant keywords, namely breast cancer therapy, Genetic Engineering, CRISPR / Cas9 Technique. The results obtained show something promising in the use of one of the genetic engineering technologies in the form of the CRISPR / Cas9 technique which is able to weaken or suppress genetic activity related to the continuity of growth and development of breast cancer cells. In conclusion, CRISPR/Cas9 technology shows promising potential in breast cancer therapy with its ability to inhibit the expression of certain genes that contribute to the growth, invasion, and metastasis of cancer cells.
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Vaishnavi, Bhad Gaurav Bhalerao Rani Deokar*. "CRISPR-CAS 9 In Gene Editing: Innovations, Applications and Ethical Challenges." International Journal of Scientific Research and Technology 1, no. 11 (2024): 59–71. https://doi.org/10.5281/zenodo.14161891.
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CRISPR-Cas9 has emerged as a revolutionary tool in gene editing, enabling precise alterations in the DNA sequence with unprecedented accuracy and efficiency. This technology leverages the natural defense mechanism of bacteria against viruses, allowing researchers to target specific genes for modification, deletion, or insertion. The potential applications of CRISPR-Cas9 are vast, ranging from therapeutic interventions for genetic disorders, cancer treatment, and viral infections to advancements in agriculture and biotechnology. Despite its promise, CRISPR-Cas9 raises significant ethical concerns. Issues such as off-target effects, the long-term safety of edited genomes, and the potential for unintended genetic consequences remain critical hurdles. Additionally, the prospect of human germline editing sparks debate around 'designer babies,' eugenics, and the moral implications of altering the human genome for non-therapeutic purposes. This review explores the latest developments in CRISPR-Cas9 technology, highlighting its transformative applications in various fields of research and medicine. It also examines the ethical challenges and regulatory frameworks needed to navigate this rapidly evolving landscape. By addressing both the potential and the pitfalls of CRISPR-Cas9, this article aims to provide a comprehensive overview of the current state of gene editing technology and its future prospects in shaping the genetic landscape.
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شحاته, د. أسماء فتحي. "كريسبر كاس 9(CRISPR Cas-9) بين دقة التطبيب ومهارة الطبيب". مجلة کلية الدراسات الإسلامية والعربية للبنات بالإسکندرية 38, № 4 (2022): 12–63. http://dx.doi.org/10.21608/bfda.2022.280035.
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Hoffmann, Mareike D., Sabine Aschenbrenner, Stefanie Grosse, et al. "Cell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins." Nucleic Acids Research 47, no. 13 (2019): e75-e75. http://dx.doi.org/10.1093/nar/gkz271.
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Abstract The rapid development of CRISPR–Cas technologies brought a personalized and targeted treatment of genetic disorders into closer reach. To render CRISPR-based therapies precise and safe, strategies to confine the activity of Cas(9) to selected cells and tissues are highly desired. Here, we developed a cell type-specific Cas-ON switch based on miRNA-regulated expression of anti-CRISPR (Acr) proteins. We inserted target sites for miR-122 or miR-1, which are abundant specifically in liver and cardiac muscle cells, respectively, into the 3′UTR of Acr transgenes. Co-expressing these with Cas9 and sgRNAs resulted in Acr knockdown and released Cas9 activity solely in hepatocytes or cardiomyocytes, while Cas9 was efficiently inhibited in off-target cells. We demonstrate control of genome editing and gene activation using a miR-dependent AcrIIA4 in combination with different Streptococcus pyogenes (Spy)Cas9 variants (full-length Cas9, split-Cas9, dCas9-VP64). Finally, to showcase its modularity, we adapted our Cas-ON system to the smaller and more target-specific Neisseria meningitidis (Nme)Cas9 orthologue and its cognate inhibitors AcrIIC1 and AcrIIC3. Our Cas-ON switch should facilitate cell-specific activity of any CRISPR–Cas orthologue, for which a potent anti-CRISPR protein is known.
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Banerjee, Sabyasachi, Shruti Gupta, Ritu Raj, Uday Kaushal, Gurpreet Kaur, and Satish Krushna Gharde. "CRISPR- Cas9 Technology: Mechanism and Its Application in The Field of Entomology." Journal of Advanced Zoology 44, no. 5 (2023): 179–93. http://dx.doi.org/10.17762/jaz.v44i5.2642.
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The field of life science research has undergone a revolution thanks to the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-associated protein (CRISPR/Cas) system, which provides a multitude of opportunities for modifying, identifying, visualising, and annotating particular DNA or RNA sequences in diverse organisms. In this technique, foreign DNA pieces, known as spacers, are inserted into CRISPR cassettes. These spacers are then transcribed into CRISPR arrays and processed to produce guide RNA (gRNA). The Cas proteins that the CRISPR arrays encode serve as the enzymatic machinery required to obtain new spacers that specifically target invasive genetic elements. Several Cas proteins, such as Cas9, Cas12, Cas13, and Cas14, have been used to create novel tools for genome engineering due to their programmable sequence specificity. The ability to manipulate and edit nucleic acid sequences in living cells from a wide variety of organisms has been made possible by these Cas variants, which have greatly advanced genetic research and the CRISPR/Cas tool. The CRISPR Cas-9 technology has applications in many areas of entomology, including the genetics of honeybees and plants that produce insecticidal compounds. CRISPR/Cas9 technology has transformed entomology by providing precise tools for gene editing and genetic manipulation in insects. This has enabled advancements in fundamental research, disease vector control, and pest management, with the potential to reduce the environmental and economic impact of insect pests in agriculture and public health.
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Briner, Alexandra E., and Rodolphe Barrangou. "Lactobacillus buchneri Genotyping on the Basis of Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Locus Diversity." Applied and Environmental Microbiology 80, no. 3 (2013): 994–1001. http://dx.doi.org/10.1128/aem.03015-13.
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ABSTRACTClustered regularly interspaced short palindromic repeats (CRISPR) in combination with associated sequences (cas) constitute the CRISPR-Cas immune system, which uptakes DNA from invasive genetic elements as novel “spacers” that provide a genetic record of immunization events. We investigated the potential of CRISPR-based genotyping ofLactobacillus buchneri, a species relevant for commercial silage, bioethanol, and vegetable fermentations. Upon investigating the occurrence and diversity of CRISPR-Cas systems inLactobacillus buchnerigenomes, we observed a ubiquitous occurrence of CRISPR arrays containing a 36-nucleotide (nt) type II-A CRISPR locus adjacent to fourcasgenes, including the universalcas1andcas2genes and the type II signature genecas9. Comparative analysis of CRISPR spacer content in 26L. buchneripickle fermentation isolates associated with spoilage revealed 10 unique locus genotypes that contained between 9 and 29 variable spacers. We observed a set of conserved spacers at the ancestral end, reflecting a common origin, as well as leader-end polymorphisms, reflecting recent divergence. Some of these spacers showed perfect identity with phage sequences, and many spacers showed homology toLactobacillusplasmid sequences. Following a comparative analysis of sequences immediately flanking protospacers that matched CRISPR spacers, we identified a novel putative protospacer-adjacent motif (PAM), 5′-AAAA-3′. Overall, these findings suggest that type II-A CRISPR-Cas systems are valuable for genotyping ofL. buchneri.
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Vasdev, Kavita. "CRISPR/Cas-9 System: Magnificent Tool for Genome Editing." International Journal of Biotechnology and Bioengineering 3, no. 9 (2017): 293–97. http://dx.doi.org/10.25141/2475-3432-2017-9.0293.
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Macena, Tharcilla Nascimento da Silva, Naiane Oliveira Santos, Matheus Almeida da Silva Gonçalves, and João Vitor de Andrade Alves. "Utilização do sistema CRISPR/CAS-9 no melhoramento vegetal:." Revista Mosaicum, no. 33 (June 10, 2021): 85–98. http://dx.doi.org/10.26893/rm.v33i33.479.
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Objetiva-se analisar as utilizações do sistema Crispr/Cas-9 no melhoramento vegetal, por meio de revisão sistemática de literatura realizada na base de dados Scopus. Elaborou-se um protocolo de pesquisa para selecionar os artigos a partir de critérios de exclusão e inclusão. Os dados desta pesquisa indicam a maior concentração de trabalhos no continente asiático, tendo a China se apresentando como principal expoente em pesquisas na área, com um foco muito grande no melhoramento de Oriza sativa e Solanum lycopersicum. Esses estudos na maioria das vezes buscam o aumento na produtividade e a tolerância a estresse abiótico e biótico isso por que há uma crescente busca por melhoria da qualidade dos grãos e melhoria da resistência a estresses diante do aquecimento climático. Palavras-chaves: Biotecnologia. Edição genética em plantas. Espécies vegetais.
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Fatima, Sana, Muhammad Muzammal, Muzammil Ahmad Khan, et al. "Crispr/Cas9 Endonucleases: A New Era of Genetic Engineering." Volume 4 Issue 2, Volume 4 Issue 2 (December 31, 2021): 29–39. http://dx.doi.org/10.34091/ajls.4.2.4.
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In modern genetic engineering, there is always development towards better ways for therapies of different diseases in the most efficient way. Genetic engineering approaches use the nucleases to cut the DNA. Meganucleases, ZFN, TALEN and CRISPR i.e. clustered regularly interspaced short palindromic repeats determine PAM sequence. These sequences are present as direct repeats that are separated by specific Spacers and have Cas genes which are present adjacent to the spacer regions. Microbe's immunity is related to the presence of CRISPR sequences, which can cleave and bind the DNA at specific sequences. CRISPR is classified into two classes that are further divided into 5 types. Most commonly used class is type 2 which work along the CRISPR associated protein called Cas 9 obtained from Streptococcus pyogene. Cas 9 is a multi-subunit protein with two nuclease domains called HNH and RuvC like domains. The presence of a smaller sequence upstream to the DNA that is to be targeted is important for specific cleavage and is called the Seed Sequence. CRISPR have many applications in genome editing and beyond genome editing. Keywords: AML; Pathogenesis; Treatment; Relapse
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Xu, Wenlina. "Research progress of gene editing technology CRISPR/Cas9 system in animal gene editing." International Journal of Veterinary Science and Research 4, no. 1 (2018): 015–19. https://doi.org/10.17352/ijvsr.000030.
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Gene editing technology, from the beginning of RNA interference (RNAi) technology to efficient developed enzyme technology, has been widely used in recent years. These efficient enzyme technologies include zinc finger nuclease (ZFN) technology, transcriptional activation-like effector nuclease (TALENs) technology, and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system (CRISPR/Cas9) technology. The CRISPR/Cas (Cas) system is a gene editing tool for DNA modification regulated by a short RNA and is a new type of genome editing tool that is faster, more efficient, and more accurate than the zinc finger nuclease and transcription activator-like effector nuclease. This article reviews the structure and function of the CRISPR/Cas system, and is aimed to outline the Cas9 design strategy, factors that affect the Cas9 gene editing efficiency, off-target detection and analysis methods, and especially the application in animal gene editing studies. Based on CRISPR/Cas9 gene editing has been successfully implemented in a variety of animals, and it is expected to become a new feasible way to establish animal models and study disease prevention in veterinary science and research.
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Siew, Wei Sheng, Yin Quan Tang, Chee Kei Kong, et al. "Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications." International Journal of Molecular Sciences 22, no. 16 (2021): 8422. http://dx.doi.org/10.3390/ijms22168422.
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Atherosclerosis represents one of the major causes of death globally. The high mortality rates and limitations of current therapeutic modalities have urged researchers to explore potential alternative therapies. The clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) system is commonly deployed for investigating the genetic aspects of Atherosclerosis. Besides, advances in CRISPR/Cas system has led to extensive options for researchers to study the pathogenesis of this disease. The recent discovery of Cas9 variants, such as dCas9, Cas9n, and xCas9 have been established for various applications, including single base editing, regulation of gene expression, live-cell imaging, epigenetic modification, and genome landscaping. Meanwhile, other Cas proteins, such as Cas12 and Cas13, are gaining popularity for their applications in nucleic acid detection and single-base DNA/RNA modifications. To date, many studies have utilized the CRISPR/Cas9 system to generate disease models of atherosclerosis and identify potential molecular targets that are associated with atherosclerosis. These studies provided proof-of-concept evidence which have established the feasibility of implementing the CRISPR/Cas system in correcting disease-causing alleles. The CRISPR/Cas system holds great potential to be developed as a targeted treatment for patients who are suffering from atherosclerosis. This review highlights the advances in CRISPR/Cas systems and their applications in establishing pathogenetic and therapeutic role of specific genes in atherosclerosis.
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Klein, Nathalie, Selina Rust, and Lennart Randau. "CRISPR-Cas-Systeme der Klasse 1: Genome Engineering und Silencing." BIOspektrum 28, no. 4 (2022): 370–73. http://dx.doi.org/10.1007/s12268-022-1775-9.
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AbstractClass 1 CRISPR-Cas systems are prevalent among prokaryotes and are characterized by effector complexes that consist of multiple Cas protein subunits. Type I systems recruit the DNA nuclease Cas3 for target DNA degradation. Type IV systems exhibit CRISPR interference in the absence of DNA cleavage. These mechanisms allow for versatile genome engineering and silencing approaches. Here, we indicate advantages and drawbacks in comparison to more commonly employed Cas9-based tools.
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Tan, Qingyuan. "CRISPR/Cas System: Mechanisms, Applications, and Limitations." BIO Web of Conferences 111 (2024): 03025. http://dx.doi.org/10.1051/bioconf/202411103025.
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The clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) gene editing system plays a role in the inhibition of immunity in many bacteria and archaea equips with various advantages such as high efficiency, diversity, and modularity. It is now widely used to improve quality and quantity of crops to satisfy global food demand. Although these prospects are tempting, deeper understanding is still required to improve their efficiency and safety. Therefore, an overview of this special system is important. In this review, the current knowledge of different types of CRISPR/Cas system as well as their mechanisms, applications in crop breeding and limitations is briefly introduced to provide fundamental understanding and guidance for future utilization.
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Ratan, Zubair Ahmed, Young-Jin Son, Mohammad Faisal Haidere, et al. "CRISPR-Cas9: a promising genetic engineering approach in cancer research." Therapeutic Advances in Medical Oncology 10 (January 1, 2018): 175883401875508. http://dx.doi.org/10.1177/1758834018755089.
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Bacteria and archaea possess adaptive immunity against foreign genetic materials through clustered regularly interspaced short palindromic repeat (CRISPR) systems. The discovery of this intriguing bacterial system heralded a revolutionary change in the field of medical science. The CRISPR and CRISPR-associated protein 9 (Cas9) based molecular mechanism has been applied to genome editing. This CRISPR-Cas9 technique is now able to mediate precise genetic corrections or disruptions in in vitro and in vivo environments. The accuracy and versatility of CRISPR-Cas have been capitalized upon in biological and medical research and bring new hope to cancer research. Cancer involves complex alterations and multiple mutations, translocations and chromosomal losses and gains. The ability to identify and correct such mutations is an important goal in cancer treatment. In the context of this complex cancer genomic landscape, there is a need for a simple and flexible genetic tool that can easily identify functional cancer driver genes within a comparatively short time. The CRISPR-Cas system shows promising potential for modeling, repairing and correcting genetic events in different types of cancer. This article reviews the concept of CRISPR-Cas, its application and related advantages in oncology.
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Dr., MuhammadUmarFarooq* Mehwish. "INHIBITIONS OF HEPATITIS C VIRUS REPLICATION WITH THE HELP OF CRISPR/CAS 9 TECHNOLOGY." INDO AMERICAN JOURNAL OF PHARMACEUTICAL SCIENCES o6, no. 05 (2019): 9578–84. https://doi.org/10.5281/zenodo.2819911.
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<em>Hepatitis c infection caused by Hepatitis c virus (HBV) is a major world-wide health problem. Current therapeutic strategies rarely eradicate HBV infections and fail to attain complete cure. There is urgent need to develop advanced</em> <em>treatment strategies to successfully remove HBV infection and eliminate hidden reservoirs of virus. Recently, the establishment of a novel RNA-guided gene editing tool known as the clustered regularly interspaced short palindromic</em> <em>repeats/CRISPR-associated nuclease 9 (CRISPR/Cas9) system, has significantly enabled site-specific mutagenesis and characterizes a very beneficial possible therapeutic means for diseases which includes extermination of invasive</em> <em>pathogens e.g. HBV. This review highlights the recent developments in the use of CRISPR/Cas9 to specifically target HBV DNA sequences for inhibition of replication of HBV and to bring mutations in viral genome, animal models.</em> <em>Benefits, restrictions and viable solutions, and proposed guidelines for forthcoming study in CRISPR/Cas9 are described to highlight the chances and challenges for curative therapy of chronic hepatitis B infection.</em> <strong>Keywords: </strong><em>Hepatitis c virus (HBV), covalently closed circular DNA (cccDNA), CRISPR/Cas9, inhibition of HBV replication, antiviral therapy.</em>
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Lone, Bilal Ahmad, Shibendra Kumar Lal Karna, Faiz Ahmad, Nerina Shahi, and Yuba Raj Pokharel. "CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering." Genetics Research International 2018 (September 18, 2018): 1–17. http://dx.doi.org/10.1155/2018/3797214.
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Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.
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Dayan, Fazli. "CRISPR Cas-9 genome editing and Islam: A religious perspective." Bangladesh Journal of Medical Science 18, no. 1 (2018): 7–13. http://dx.doi.org/10.3329/bjms.v18i1.39540.
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Background: Certainly, the ultimate aim of Islamic law is to “protect human life” either through mitigation of hardship or recognition of public interests reckons biomedical innovations allowable where-if cling by ethical, moral and legal principles. Assertively, if–CRISPR Cas- 9 genome editing–methods based on the guided principles of Islamic law and jurisprudence, as “harm has to be redressed” can be justified keeping in view the human dignity, honor and prestige. Hence, newer technologies can be adopted because “necessity renders prohibited things as permissible” with certain caveats. Arguably those who consider it as an evil must think over that “in the presence of two evils, the one whose injury is greater is avoided by the commission of the lesser”.
 Conclusion: Therefore if Cas-9 based method leaning towards evils, even then it can be acceptable in case where an atypical germ-line sequence can affect the next generation, which is indeed a great evil, and “the lesser of evils is preferred over the greater one” renders it permissible with a view it might enhance human health and living standard. Conversely, curing a minor disease if causing another equal infirmity or greater should be rendered forbidden as “harm cannot be removed by harm”, then, “a greater harm can be removed by a lesser one” germ-line editing/alteration in severe cases will be allowed on the basis of necessity.
 Bangladesh Journal of Medical Science Vol.18(1) 2019 p.7-13
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Mengstie, Misganaw Asmamaw, and Belay Zawdie Wondimu. "Mechanism and Applications of CRISPR/Cas-9-Mediated Genome Editing." Biologics: Targets and Therapy Volume 15 (August 2021): 353–61. http://dx.doi.org/10.2147/btt.s326422.
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Santoso, Tri Joko, Atmitri Sisharmini, Aniversari Apriana, Kristianto Nugroho, and Alberta Dinar Ambarwati. "Strategy to select the CRISPR/Cas9 target sequences of the sucrose-phosphate synthase gene for assembling an efficient gRNA spacer in shallot." BIO Web of Conferences 127 (2024): 01008. http://dx.doi.org/10.1051/bioconf/202412701008.
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Bacterial leaf blight (BLB), caused by Xanthomonas axonopodis pv. Allii, is a newly identified pathogen that infects shallots. This disease can lead to yield losses ranging from 20% to 100%. The genome editing system via CRISPR/Cas-9 is a technology that can be used to modify susceptibility genes accurately and precisely. The target gene for CRISPR/Cas-9 genome editing system to develop a BLB-resistant shallot is the sucrose-phosphate synthase (SPS). One of the critical factors in the CRSPR/Cas-9 genome editing includes the preparation of single guide RNA (sgRNA) design and construction of it into an expression plasmid vector. The study aimed to develop strategies for selecting the SPS gene’s CRISPR/Cas9 target sequences to assemble an efficient gRNA spacer in shallot. The gRNA design of the SPS gene was carried out using the software at http://crispor.tefor.net/. The efficiency of sgRNAs is then filtered and predicted based on GC contents, the last four nucleotides, secondary RNA structure, and stem loops. The construction of the CRISPR/Cas9 module was carried out using the Golden Gate method. The results showed that based on the selection of target gRNA sequences in the SPS gene, one of the best gRNAs, gRNA-54/forw, has been produced for use in SPS gene editing research. The best-selected gRNA of the SPS gene was then successfully inserted into the CRISPR/Cas9 pRGEB32 cassette vector after verification using DNA sequencing analysis. Based on this result, the pRGEB32_gRNA-AcSPS construct is ready to be introduced into shallot to develop a BLB-resistant shallot variety.
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Kalidoss, Karthik. "CRISPR-Cas Genome Editing Tool: Mechanisms of Pathogen Resistance Plants – Review." Journal of Horticulture and Plant Research 7 (August 2019): 69–80. http://dx.doi.org/10.18052/www.scipress.com/jhpr.7.69.
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In recent years, the CRISPR-Cas system is most familiar and advance genome editing tool in modern biological research. The genome editing tool used in various biological researchers worldwide in past years has witnessed exposure site-directed mutagenesis modification methods zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), Meganucleases and CRISPR-Cas9(associated proteins 9). CRISPR-Cas genome editing technology to ease design and implement, more flexible and less expensive. Plants are affected two types of stresses like biotic and abiotic. Abiotic occurs naturally temperature or wind, sunlight depend upon on the environmental conditions. Biotic stress is caused by pathogens of virus, fungi, bacteria, etc. This review to focus on the recent advance of plant protection use CRISPR-Cas system mechanism of disease resistant plants in past and current trends of research. A short overview of the experimental methodology for Beet Curly Top Virus (BCTV) disease and Magnaporthe oryzae fungus infection cause rice blast disease resistance mechanisms will be discussed. Furthermore, the need developments of this genome editing tool in future.
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Yun, Dayoung, and Cheulhee Jung. "MiRNA-Responsive CRISPR-Cas System via a DNA Regulator." Biosensors 13, no. 11 (2023): 975. http://dx.doi.org/10.3390/bios13110975.
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Clustered regularly interspaced short palindromic repeats (CRISPR)- CRISPR-associated protein 9 (Cas9) genome editing technology is widely used for gene editing because it provides versatility in genetic manipulation. Several methods for regulating CRISPR activity already exist for accurate editing, but these require complex engineering. Thus, a simple and convenient regulatory system is required. In this study, we devised a CRISPR activation system using a DNA regulator that can be activated by miRNAs. The designed regulator was divided into two parts. The inhibition component consisted of the protospacer-adjacent motif (PAM) and seed sequence, which are important for Cas9 target recognition and bind to the ribonucleoprotein (RNP) complex for inhibition. The miRNA recognition component has a single-stranded toehold DNA for target miRNA binding and a partial double-stranded DNA complementary to the remaining miRNA sequence. In the presence of target miRNAs, the structure of the regulator is disrupted by the miRNAs, leading to its dissociation from the RNP complex and subsequent restoration of CRISPR activity. This method is easy to design and can be applied to various miRNAs via simple sequence manipulation. Therefore, this strategy provides a general platform for controlled genome editing.
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Singh, Desh D., Ravi Verma, Piyush Parimoo, et al. "Potential Therapeutic Relevance of CRISPR/Cas9 Guided Epigenetic Regulations for Neuropsychiatric Disorders." Current Topics in Medicinal Chemistry 21, no. 10 (2021): 878–94. http://dx.doi.org/10.2174/1568026621666210317154502.
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Brain function activity is regulated by several mechanisms of genetic and epigenetic factors such as histone modelling, DNA methylation, and non-coding RNA. Alterations in these regulatory mechanisms affect the normal development of neurons that causes Neuropsychiatric Disorders (ND). However, it is required to analyse the functional significance of neuropsychiatric disorders associated with a molecular mechanism to bring about therapeutic advances in early diagnosis and treatment of the patients. The CRISPR/Cas 9 (Clustered Regularly Interspaced Short Palindromic Repeats) genome editing tools have revolutionized multiple genome and epigenome manipulation targets the same time. This review discussed the possibilities of using CRISPR/Cas 9 tools during molecular mechanism in the ND as a therapeutic approach to overcome ND that is caused due to genetic and epigenetic abnormalities.
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Bhagwat, Amrita C., Amrita M. Patil, and Sunil D. Saroj. "CRISPR/Cas 9-Based Editing in the Production of Bioactive Molecules." Molecular Biotechnology 64, no. 3 (2021): 245–51. http://dx.doi.org/10.1007/s12033-021-00418-4.
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Gupta, N., K. Polkoff, L. Qiao, K. Cheng, and J. Piedrahita. "200 Developing exosomes as a mediator for CRISPR/Cas-9 delivery." Reproduction, Fertility and Development 31, no. 1 (2019): 225. http://dx.doi.org/10.1071/rdv31n1ab200.
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CRISPR/Cas systems present a powerful gene-editing tool with the potential for widespread therapeutic use; however, current methods of in vivo delivery such as adeno-associated viruses (AAV) may stimulate an immune response, creating the need for an alternative for delivery of CRISPR/Cas9. Exosomes are small vesicles that are released by cells and serve as a delivery system for RNA, proteins, and various molecules to other cells. The focus of this project was to use exosomes as a delivery system for Cas9, exploiting their high uptake by target cells and their ability to avoid the immune system in vivo. Porcine fetal fibroblasts (PFF) were grown to 80% confluency; after 48h, exosomes were isolated and concentrated from conditioned media by filtration with a 0.22-μm filter followed by 100-kDa molecular weight cutoff filter. Transmission electron microscopy, Western blotting for presence of CD81, and an uptake assay for exosomes stained with the lipophilic dye DiI (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA) were used to characterise isolated exosomes, and average particle size was evaluated by NanoSight (Salisbury, United Kingdom). After characterisation, exosomes were loaded with Cas9 (PNA Bio, Newbury Park, CA, USA) using sonication, incubation with saponin, or extrusion. For each method of loading, 1.0×1011 exosomes and 500ng of Cas9 were used. For sonication, exosomes and Cas9 were sonicated 4 times: 4s on/2s off, left on ice for 2min, and then repeated for 4 more cycles. Loaded exosomes were then incubated at 37°C for 20min. For incubation with saponin, 100μL of 0.6% saponin solution was made in PBS, mixed with exosomes and Cas9, and then incubated on a shaker at 800 rpm for 20min. For extrusion, exosomes and Cas9 were extruded (Avanti Polar Lipids, Alabaster, AL, USA) 10, 15, or 20 times through a 0.22-μm filter. To evaluate efficiency of Cas9 loading into exosomes, loaded exosome samples were split in half, with one-half receiving a proteinase K digest (100μg mL−1) to remove free Cas9 and the other receiving no treatment. Proteinase K-treated and untreated samples were then compared side by side on Western blot staining for Cas9. ImageJ software (National Institutes for Health, Bethesda, MD, USA) was used to quantify band intensity and loading efficiency. With optimal conditions, our preliminary results show loading efficiency for sonication and saponin to be 16.7 and 19.2%, respectively, whereas loading by extrusion was undetectable. For CRISPR/Cas targeting, transgenic PFF carrying one copy of H2B-GFP were used to test delivery of ribonucleotide protein complex (RNP). To verify efficiency of the guide (g)RNA targeting green fluorescent protein (GFP), cells were nucleofected with Cas9 and gRNA. The DNA was extracted, PCR amplified, and sequenced (Eton Bioscience, San Diego, CA, USA) and then evaluated for indels with TIDE, resulting in a 53.2% cleavage efficiency. Next, exosomes will be loaded with RNP to knockout GFP in H2B-GFP cells, and targeting efficiency will be evaluated by flow cytometry and TIDE. We hypothesise that based on loading efficiency and target cell uptake, exosomes will present a safe and efficient method for in vitro and in vivo delivery of Cas9. The financial support of the Comparative Medicine Institute is gratefully acknowledged.
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Ebrahimi, Saeedeh, Ali Teimoori, Hashem Khanbabaei, and Maryam Tabasi. "Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome." Reviews in Medical Virology 29, no. 1 (2018): e2009. http://dx.doi.org/10.1002/rmv.2009.
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Tripathi, Manoj Kumar, Pankaj kumar, Bosco Jose, Sukanta Mondal, Pranay kumar K та Kamal Sarma. "CRISPR-CAS 9 and its application as therapeutics for β-haemoglobinopathies". Exploratory Animal and Medical Research 13, № 1 (2023): 08–15. http://dx.doi.org/10.52635/eamr/13.1.8-15.
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Chandran, Sarankumar, Valarmathi Muthu, Tharshenee Umapathy, Sowmiya Jayakumar, and Sindhuja Chokkalingam. "CRISPR / Cas 9 assisted genome editing technology for the improvement of Horticultural crops." Journal of Phytopharmacology 12, no. 2 (2023): 127–34. http://dx.doi.org/10.31254/phyto.2023.12110.
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Horticultural crops produce a wide range of useful goods for humans. There has been an increase in research focus on horticulture crop enhancement, particularly in terms of production and quality. The use of genome editing to enhance horticulture crops has seen a sharp rise in recent years due to the advancement and benefits of genome-editing technology. Here, we provide a brief overview of the various genome-editing techniques applied in horticulture research, with a particular emphasis on CRISPR/CRISPR-associated 9 (Cas9)-mediated genome editing. We also provide an overview of recent developments in the use of genome editing to enhance horticulture crops. Breeding and the rapidly growing field of genome editing will significantly boost the quantity and quality of horticulture crops.
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Değirmenci, Laura, Dietmar Geiger, Fábio Luiz Rogé Ferreira, et al. "CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees." Chemical Senses 45, no. 8 (2020): 655–66. http://dx.doi.org/10.1093/chemse/bjaa063.
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Abstract Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose, and fructose are the main components of plant nectars. Intriguingly, honeybees express only 3 putative sugar receptors (AmGr1, AmGr2, and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioral change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterization in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.
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Karpov, Dmitry S., Natalia A. Demidova, Kirill A. Kulagin, et al. "Complete and Prolonged Inhibition of Herpes Simplex Virus Type 1 Infection In Vitro by CRISPR/Cas9 and CRISPR/CasX Systems." International Journal of Molecular Sciences 23, no. 23 (2022): 14847. http://dx.doi.org/10.3390/ijms232314847.
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Almost all people become infected with herpes viruses, including herpes simplex virus type 1 (HSV-1), during their lifetime. Typically, these viruses persist in a latent form that is resistant to all available antiviral medications. Under certain conditions, such as immunosuppression, the latent forms reactivate and cause disease. Moreover, strains of herpesviruses that are drug-resistant have rapidly emerged. Therefore, it is important to develop alternative methods capable of eradicating herpesvirus infections. One promising direction is the development of CRISPR/Cas systems for the therapy of herpesvirus infections. We aimed to design a CRISPR/Cas system for relatively effective long-term and safe control of HSV-1 infection. Here, we show that plasmids encoding the CRISPR/Cas9 system from Streptococcus pyogenes with a single sgRNA targeting the UL30 gene can completely suppress HSV-1 infection of the Vero cell line within 6 days and provide substantial protection within 9 days. For the first time, we show that CRISPR/CasX from Deltaproteobacteria with a single guide RNA against UL30 almost completely suppresses HSV-1 infection of the Vero cell line for 3 days and provides substantial protection for 6 days. We also found that the Cas9 protein without sgRNAs attenuates HSV-1 infection. Our results show that the developed CRISPR/Cas systems are promising therapeutic approaches to control HSV-1 infections.
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Serajian, Sahar, Ehsan Ahmadpour, Sonia M. Rodrigues Oliveira, Maria de Lourdes Pereira, and Siamak Heidarzadeh. "CRISPR-Cas Technology: Emerging Applications in Clinical Microbiology and Infectious Diseases." Pharmaceuticals 14, no. 11 (2021): 1171. http://dx.doi.org/10.3390/ph14111171.
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Through the years, many promising tools for gene editing have been developed including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), CRISPR-associated protein 9 (Cas9), and homing endonucleases (HEs). These novel technologies are now leading new scientific advancements and practical applications at an inimitable speed. While most work has been performed in eukaryotes, CRISPR systems also enable tools to understand and engineer bacteria. The increase in the number of multi-drug resistant strains highlights a necessity for more innovative approaches to the diagnosis and treatment of infections. CRISPR has given scientists a glimmer of hope in this area that can provide a novel tool to fight against antimicrobial resistance. This system can provide useful information about the functions of genes and aid us to find potential targets for antimicrobials. This paper discusses the emerging use of CRISPR-Cas systems in the fields of clinical microbiology and infectious diseases with a particular emphasis on future prospects.
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Hao, Min, Zhaoguan Wang, Hongyan Qiao, Peng Yin, Jianjun Qiao, and Hao Qi. "Dynamic Genome Editing Using In Vivo Synthesized Donor ssDNA in Escherichia coli." Cells 9, no. 2 (2020): 467. http://dx.doi.org/10.3390/cells9020467.
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As a key element of genome editing, donor DNA introduces the desired exogenous sequence while working with other crucial machinery such as CRISPR-Cas or recombinases. However, current methods for the delivery of donor DNA into cells are both inefficient and complicated. Here, we developed a new methodology that utilizes rolling circle replication and Cas9 mediated (RC-Cas-mediated) in vivo single strand DNA (ssDNA) synthesis. A single-gene rolling circle DNA replication system from Gram-negative bacteria was engineered to produce circular ssDNA from a Gram-positive parent plasmid at a designed sequence in Escherichia coli. Furthermore, it was demonstrated that the desired linear ssDNA fragment could be cut out using CRISPR-associated protein 9 (CRISPR-Cas9) nuclease and combined with lambda Red recombinase as donor for precise genome engineering. Various donor ssDNA fragments from hundreds to thousands of nucleotides in length were synthesized in E. coli cells, allowing successive genome editing in growing cells. We hope that this RC-Cas-mediated in vivo ssDNA on-site synthesis system will be widely adopted as a useful new tool for dynamic genome editing.
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Markusková, Barbora, Aneta Lichvariková, Tomáš Szemes, Janka Koreňová, Tomáš Kuchta, and Hana Drahovská. "Genome analysis of lactic acid bacterial strains selected as potential starters for traditional Slovakian bryndza cheese." FEMS Microbiology Letters 366, Supplement_1 (2018): i3—i9. http://dx.doi.org/10.1093/femsle/fny257s.
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ABSTRACT Genomes of 21 strains of lactic acid bacteria isolated from Slovakian traditional cheeses were sequenced on an Illumina MiSeq platform. Subsequently, they were analysed regarding taxonomic classification, presence of genes encoding defence systems, antibiotic resistance and production of biogenic amines. Thirteen strains were found to carry genes encoding at least one bacteriocin, 18 carried genes encoding at least one restriction–modification system, all strains carried 1–6 prophages and 9 strains had CRISPR-Cas systems. CRISPR-Cas type II-A was the most common, containing 0–24 spacers. Only 10% spacers were found to be homological to known bacteriophage or plasmid sequences in databases. Two Enterococcus faecium strains and a Lactococcus lactis strain carried antibiotic resistance genes. Genes encoding for ornithine decarboxylase were detected in four strains and genes encoding for agmatine deiminase were detected in four strains. Lactobacillus paraplantarum 251 L appeared to be the most interesting strain, as it contained genes encoding for two bacteriocins, a restriction–modification system, two CRISPR-Cas systems, four prophages and no genes connected with antibiotic resistance or production of biogenic amines.
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Kaupbayeva, Bibifatima, Andrey Tsoy, Yuliya Safarova (Yantsen), et al. "Unlocking Genome Editing: Advances and Obstacles in CRISPR/Cas Delivery Technologies." Journal of Functional Biomaterials 15, no. 11 (2024): 324. http://dx.doi.org/10.3390/jfb15110324.
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CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats associated with protein 9) was first identified as a component of the bacterial adaptive immune system and subsequently engineered into a genome-editing tool. The key breakthrough in this field came with the realization that CRISPR/Cas9 could be used in mammalian cells to enable transformative genetic editing. This technology has since become a vital tool for various genetic manipulations, including gene knockouts, knock-in point mutations, and gene regulation at both transcriptional and post-transcriptional levels. CRISPR/Cas9 holds great potential in human medicine, particularly for curing genetic disorders. However, despite significant innovation and advancement in genome editing, the technology still possesses critical limitations, such as off-target effects, immunogenicity issues, ethical considerations, regulatory hurdles, and the need for efficient delivery methods. To overcome these obstacles, efforts have focused on creating more accurate and reliable Cas9 nucleases and exploring innovative delivery methods. Recently, functional biomaterials and synthetic carriers have shown great potential as effective delivery vehicles for CRISPR/Cas9 components. In this review, we attempt to provide a comprehensive survey of the existing CRISPR-Cas9 delivery strategies, including viral delivery, biomaterials-based delivery, synthetic carriers, and physical delivery techniques. We underscore the urgent need for effective delivery systems to fully unlock the power of CRISPR/Cas9 technology and realize a seamless transition from benchtop research to clinical applications.
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Selvam, Kasturi, Mohamad Ahmad Najib, Muhammad Fazli Khalid, Mehmet Ozsoz, and Ismail Aziah. "CRISPR-Cas Systems-Based Bacterial Detection: A Scoping Review." Diagnostics 12, no. 6 (2022): 1335. http://dx.doi.org/10.3390/diagnostics12061335.
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Recently, CRISPR-Cas system-based assays for bacterial detection have been developed. The aim of this scoping review is to map existing evidence on the utilization of CRISPR-Cas systems in the development of bacterial detection assays. A literature search was conducted using three databases (PubMed, Scopus, and Cochrane Library) and manual searches through the references of identified full texts based on a PROSPERO-registered protocol (CRD42021289140). Studies on bacterial detection using CRISPR-Cas systems that were published before October 2021 were retrieved. The Critical Appraisal Skills Programme (CASP) qualitative checklist was used to assess the risk of bias for all the included studies. Of the 420 studies identified throughout the search, 46 studies that met the inclusion criteria were included in the final analysis. Bacteria from 17 genera were identified utilising CRISPR-Cas systems. Most of the bacteria came from genera such as Staphylococcus, Escherichia, Salmonella, Listeria, Mycobacterium and Streptococcus. Cas12a (64%) is the most often used Cas enzyme in bacterial detection, followed by Cas13a (13%), and Cas9 (11%). To improve the signal of detection, 83% of the research exploited Cas enzymes’ trans-cleavage capabilities to cut tagged reporter probes non-specifically. Most studies used the extraction procedure, whereas only 17% did not. In terms of amplification methods, isothermal reactions were employed in 66% of the studies, followed by PCR (23%). Fluorescence detection (67%) was discovered to be the most commonly used method, while lateral flow biosensors (13%), electrochemical biosensors (11%), and others (9%) were found to be less commonly used. Most of the studies (39) used specific bacterial nucleic acid sequences as a target, while seven used non-nucleic acid targets, including aptamers and antibodies particular to the bacteria under investigation. The turnaround time of the 46 studies was 30 min to 4 h. The limit of detection (LoD) was evaluated in three types of concentration, which include copies per mL, CFU per mL and molarity. Most of the studies used spiked samples (78%) rather than clinical samples (22%) to determine LoD. This review identified the gap in clinical accuracy evaluation of the CRISPR-Cas system in bacterial detection. More research is needed to assess the diagnostic sensitivity and specificity of amplification-free CRISPR-Cas systems in bacterial detection for nucleic acid-based tests.
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Zhu, Haocheng, Chao Li, and Caixia Gao. "Applications of CRISPR–Cas in agriculture and plant biotechnology." Nature Reviews Molecular Cell Biology 21, no. 11 (2020): 661–77. http://dx.doi.org/10.1038/s41580-020-00288-9.
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Torres, Cristiane Batista Bezerra, and Wagner Soares Pessoa. "Células-tronco pluripotentes induzidas e edição de genes: avanços tecnológicos da pesquisa em medicina regenerativa e terapia gênica." Jornal Interdisciplinar de Biociências 3, no. 1 (2018): 56. http://dx.doi.org/10.26694/jibi.v3i1.6258.
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A reprogramação gênica de células diferenciadas permitiu a obtenção de células-tronco pluripotentes induzidas (induced pluripotent stem cells – iPSCs) que não apresentam os questionamentos éticos que envolvem as células-tronco embrionárias e nem o risco de rejeição imunológica. A tecnologia do Conjunto de Repetições Palindrômicas Regularmente Espaçadas com Nuclease Associada 9 (CRISPR-Cas9), permite a correção de defeitos genéticos. O presente estudo tem por objetivo revisar as principais questões metodológicas relacionadas às iPSCs e o CRISPR-Cas 9. Realizou-se uma busca eletrônica nas bases de dados LILACS, MEDLINE, PubMed e SciELO, por meio das expressões “induced pluripotent stem cells ” e “CRISPR Cas9”. As iPSCs podem ser expandidas em cultura e diferenciadas em qualquer célula do corpo, consistindo em um modelo útil para a edição de genes com o CRISPR-Cas9, abrindo novas perspectivas na pesquisa em medicina regenerativa e terapia gênica.
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Dayan, Fazli. "Ethico-legal aspects of CRISPR Cas-9 genome editing: A balanced approach." Bangladesh Journal of Medical Science 19, no. 1 (2019): 11–16. http://dx.doi.org/10.3329/bjms.v19i1.43869.
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Notably, reproductive technology and its applications in human subject are not only debatable ethically but also religiously, where objections are raised by the contemporary scholars and specialists of the field on CRISPR Cas-9 due to its potential application for the genome editing. This does however generated a dialogue both in religion and modern ethico-legal world regime. Some contemporary bioethicists are of the view that this technology is one of those issues which have the most complex ethical concerns, fearing that this technology could transforms with the expectations and ambitions about human control over the biological world. Consequently, this is an area of anxiety not only for the bioethicists but also for the theologians. Thus it needs proper investigation, as it is not solely a scientific innovation, but in fact an ethical, legal and biomedical issue.
 Bangladesh Journal of Medical Science Vol.19(1) 2020 p.11-16
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Lv, Wei, Tao Li, Shanshan Wang, et al. "The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer." Technology in Cancer Research & Treatment 20 (January 2021): 153303382110452. http://dx.doi.org/10.1177/15330338211045206.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was originally discovered in prokaryotes and functions as part of the adaptive immune system. The experimental research of many scholars, as well as scientific and technological advancements, has allowed prokaryote-derived CRISPR/Cas genome-editing systems to transform our ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in the living cells of diverse species. Through modern genetic engineering editing technology and high-throughput gene sequencing, we can edit and splice covalently closed circular DNA to silence it, and correct the mutation and deletion of liver cancer genes to achieve precise in situ repair of defective genes and prohibit viral infection or replication. Such manipulations do not destroy the structure of the entire genome and facilitate the cure of diseases. In this review, we discussed the possibility that CRISPR/Cas could be used as a treatment for patients with liver cancer caused by hepatitis B virus infection, and reviewed the challenges incurred by this effective gene-editing technology.
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Lv, Wei, Tao Li, Shanshan Wang, et al. "The Application of the CRISPR/Cas9 System in the Treatment of Hepatitis B Liver Cancer." Technology in Cancer Research & Treatment 20 (January 2021): 153303382110452. http://dx.doi.org/10.1177/15330338211045206.
Full textAbstract:
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system was originally discovered in prokaryotes and functions as part of the adaptive immune system. The experimental research of many scholars, as well as scientific and technological advancements, has allowed prokaryote-derived CRISPR/Cas genome-editing systems to transform our ability to manipulate, detect, image, and annotate specific DNA and RNA sequences in the living cells of diverse species. Through modern genetic engineering editing technology and high-throughput gene sequencing, we can edit and splice covalently closed circular DNA to silence it, and correct the mutation and deletion of liver cancer genes to achieve precise in situ repair of defective genes and prohibit viral infection or replication. Such manipulations do not destroy the structure of the entire genome and facilitate the cure of diseases. In this review, we discussed the possibility that CRISPR/Cas could be used as a treatment for patients with liver cancer caused by hepatitis B virus infection, and reviewed the challenges incurred by this effective gene-editing technology.