Relevant bibliographies by topics / CRISPR-Cas Systems

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  3. Books
  4. Book chapters
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Academic literature on the topic 'CRISPR-Cas Systems'

Author: Grafiati
Published: 4 June 2021
Last updated: 18 May 2024

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Journal articles on the topic "CRISPR-Cas Systems"

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Roslan, Rozieffa, Peer Mohamed Abdul, and Jamaliah Md Jahim. "Endogenous CRISPR/Cas Systems Prediction: A Glimpse towards Harnessing CRISPR/ Cas Machineries for Genetic Engineering." Jurnal Kejuruteraan si1, no. 7 (November 30, 2018): 1–9. http://dx.doi.org/10.17576/jkukm-2018-si1(7)-01.

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Genetic engineering field has become an imperative approach for enhancement of various bioproducts yield and productivity; and found extended applications in gene therapy, nanotechnology, as well as industrial microbiology. Modern genetic engineering tool CRISPR/Cas system, specifically the Type II system from Streptococcus pyogenes, is gaining traction in recent years and being utilized to engineer novel strains to overproduce primary fermentation product of interest. Employing this technology for non-model microorganism such as Clostridium spp is still restricted due to several limitations such as inadequate genome information, resistance against transformation, low plasmid replication, and the ability for gene expression. The prediction of CRISPR/Cas systems in microbial genomes is fundamentally the initial step towards exploitation of this technology to engineer Clostridium spp. In this study, we demonstrate a simple yet effective method to predict component of endogenous CRISPR/Cas systems, using Clostridium spp genomes as a proof-of-concept. We identified the “real” CRISPR array together with the cas gene operon consist of Type I B signature proteins in Clostridium pasteurianum which is in agreement with the previous report, implying that this strategy generates reliable CRISPR/Cas systems prediction. Thus, this provides a glimpse on how bioinformatics and biocomputational tools can be utilized to overcome barriers in genetic engineering.
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Shmakov, Sergey A., Kira S. Makarova, Yuri I. Wolf, Konstantin V. Severinov, and Eugene V. Koonin. "Systematic prediction of genes functionally linked to CRISPR-Cas systems by gene neighborhood analysis." Proceedings of the National Academy of Sciences 115, no. 23 (May 21, 2018): E5307—E5316. http://dx.doi.org/10.1073/pnas.1803440115.

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The CRISPR-Cas systems of bacterial and archaeal adaptive immunity consist of direct repeat arrays separated by unique spacers and multiple CRISPR-associated (cas) genes encoding proteins that mediate all stages of the CRISPR response. In addition to the relatively small set of core cas genes that are typically present in all CRISPR-Cas systems of a given (sub)type and are essential for the defense function, numerous genes occur in CRISPR-cas loci only sporadically. Some of these have been shown to perform various ancillary roles in CRISPR response, but the functional relevance of most remains unknown. We developed a computational strategy for systematically detecting genes that are likely to be functionally linked to CRISPR-Cas. The approach is based on a “CRISPRicity” metric that measures the strength of CRISPR association for all protein-coding genes from sequenced bacterial and archaeal genomes. Uncharacterized genes with CRISPRicity values comparable to those of cas genes are considered candidate CRISPR-linked genes. We describe additional criteria to predict functionally relevance for genes in the candidate set and identify 79 genes as strong candidates for functional association with CRISPR-Cas systems. A substantial majority of these CRISPR-linked genes reside in type III CRISPR-cas loci, which implies exceptional functional versatility of type III systems. Numerous candidate CRISPR-linked genes encode integral membrane proteins suggestive of tight membrane association of CRISPR-Cas systems, whereas many others encode proteins implicated in various signal transduction pathways. These predictions provide ample material for improving annotation of CRISPR-cas loci and experimental characterization of previously unsuspected aspects of CRISPR-Cas system functionality.
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Zhuang, Xiwei, Xueqiong Yang, Bo Cao, Haiming Sun, Xiaoyan Lv, Chijia Zeng, Fugang Li, et al. "Review—CRISPR/Cas Systems: Endless Possibilities for Electrochemical Nucleic Acid Sensors." Journal of The Electrochemical Society 169, no. 3 (March 1, 2022): 037522. http://dx.doi.org/10.1149/1945-7111/ac5cec.

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The CRISPR/Cas system has gained enormous attention for its excellent gene-editing capabilities. In recent years, the reported trans-cleavage activity of some Cas proteins, including Cas12, Cas13 and Cas14, has given the CRISPR/Cas system an increasingly powerful molecular diagnostic ability. When the CRISPR/Cas system is introduced into the field of electrochemical (EC) biosensor, it confers the high specificity to distinguish single base mismatches of nucleic acid, excellent sensitivity with the limit of detection as low as attomole range, and well meets the point-of-care testing (POCT) requirements of nucleic acid testing (NAT). In this review, we have briefly introduced the history and inherent advantages of the CRISPR/Cas system. The EC sensing platforms based on CRISPR/Cas systems have been compared with the classical fluorescence and colorimetric platforms. And the isothermal amplification strategies suitable for CRISPR/Cas system have been summarized. After that, we have highlighted the application of EC biosensor based on CRISPR/Cas system (EC-CRISPR) in the detection and identification of cancers, bacteria and viruses. Finally, the future prospects of EC-CRISPR have been proposed.
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Li, Ming, Luyao Gong, Feiyue Cheng, Haiying Yu, Dahe Zhao, Rui Wang, Tian Wang, et al. "Toxin-antitoxin RNA pairs safeguard CRISPR-Cas systems." Science 372, no. 6541 (April 29, 2021): eabe5601. http://dx.doi.org/10.1126/science.abe5601.

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CRISPR-Cas systems provide RNA-guided adaptive immunity in prokaryotes. We report that the multisubunit CRISPR effector Cascade transcriptionally regulates a toxin-antitoxin RNA pair, CreTA. CreT (Cascade-repressed toxin) is a bacteriostatic RNA that sequesters the rare arginine tRNAUCU (transfer RNA with anticodon UCU). CreA is a CRISPR RNA–resembling antitoxin RNA, which requires Cas6 for maturation. The partial complementarity between CreA and the creT promoter directs Cascade to repress toxin transcription. Thus, CreA becomes antitoxic only in the presence of Cascade. In CreTA-deleted cells, cascade genes become susceptible to disruption by transposable elements. We uncover several CreTA analogs associated with diverse archaeal and bacterial CRISPR-cas loci. Thus, toxin-antitoxin RNA pairs can safeguard CRISPR immunity by making cells addicted to CRISPR-Cas, which highlights the multifunctionality of Cas proteins and the intricate mechanisms of CRISPR-Cas regulation.
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Shehreen, Saadlee, Te-yuan Chyou, Peter C. Fineran, and Chris M. Brown. "Genome-wide correlation analysis suggests different roles of CRISPR-Cas systems in the acquisition of antibiotic resistance genes in diverse species." Philosophical Transactions of the Royal Society B: Biological Sciences 374, no. 1772 (March 25, 2019): 20180384. http://dx.doi.org/10.1098/rstb.2018.0384.

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CRISPR-Cas systems are widespread in bacterial and archaeal genomes, and in their canonical role in phage defence they confer a fitness advantage. However, CRISPR-Cas may also hinder the uptake of potentially beneficial genes. This is particularly true under antibiotic selection, where preventing the uptake of antibiotic resistance genes could be detrimental. Newly discovered features within these evolutionary dynamics are anti-CRISPR genes, which inhibit specific CRISPR-Cas systems. We hypothesized that selection for antibiotic resistance might have resulted in an accumulation of anti-CRISPR genes in genomes that harbour CRISPR-Cas systems and horizontally acquired antibiotic resistance genes. To assess that question, we analysed correlations between the CRISPR-Cas, anti-CRISPR and antibiotic resistance gene content of 104 947 reference genomes, including 5677 different species. In most species, the presence of CRISPR-Cas systems did not correlate with the presence of antibiotic resistance genes. However, in some clinically important species, we observed either a positive or negative correlation of CRISPR-Cas with antibiotic resistance genes. Anti-CRISPR genes were common enough in four species to be analysed. In Pseudomonas aeruginosa , the presence of anti-CRISPRs was associated with antibiotic resistance genes. This analysis indicates that the role of CRISPR-Cas and anti-CRISPRs in the spread of antibiotic resistance is likely to be very different in particular pathogenic species and clinical environments. This article is part of a discussion meeting issue ‘The ecology and evolution of prokaryotic CRISPR-Cas adaptive immune systems’.
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Yang, Shanshan, Jian Huang, and Bifang He. "CASPredict: a web service for identifying Cas proteins." PeerJ 9 (July 30, 2021): e11887. http://dx.doi.org/10.7717/peerj.11887.

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Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins constitute the CRISPR-Cas systems, which play a key role in prokaryote adaptive immune system against invasive foreign elements. In recent years, the CRISPR-Cas systems have also been designed to facilitate target gene editing in eukaryotic genomes. As one of the important components of the CRISPR-Cas system, Cas protein plays an irreplaceable role. The effector module composed of Cas proteins is used to distinguish the type of CRISPR-Cas systems. Effective prediction and identification of Cas proteins can help biologists further infer the type of CRISPR-Cas systems. Moreover, the class 2 CRISPR-Cas systems are gradually applied in the field of genome editing. The discovery of Cas protein will help provide more candidates for genome editing. In this paper, we described a web service named CASPredict (http://i.uestc.edu.cn/caspredict/cgi-bin/CASPredict.pl) for identifying Cas proteins. CASPredict first predicts Cas proteins based on support vector machine (SVM) by using the optimal dipeptide composition and then annotates the function of Cas proteins based on the hmmscan search algorithm. The ten-fold cross-validation results showed that the 84.84% of Cas proteins were correctly classified. CASPredict will be a useful tool for the identification of Cas proteins, or at least can play a complementary role to the existing methods in this area.
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Burmistrz, Michał, and Krzysztof Pyrc. "CRISPR-Cas Systems in Prokaryotes." Polish Journal of Microbiology 64, no. 3 (September 18, 2015): 193–202. http://dx.doi.org/10.5604/01.3001.0009.2114.

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Prokaryotic organisms possess numerous strategies that enable survival in hostile conditions. Among others, these conditions include the invasion of foreign nucleic acids such as bacteriophages and plasmids. The clustered regularly interspaced palindromic repeats-CRISPR-associated proteins (CRISPR-Cas) system provides the majority of bacteria and archaea with adaptive and hereditary immunity against this threat. This mechanism of immunity is based on short fragments of foreign DNA incorporated within the hosts genome. After transcription, these fragments guide protein complexes that target foreign nucleic acids and promote their degradation. The aim of this review is to summarize the current status of CRISPR-Cas research, including the mechanisms of action, the classification of different types and subtypes of these systems, and the development of new CRISPR-Cas-based molecular biology tools.
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Li, Junwei, Yuexia Wang, Bin Wang, Juan Lou, Peng Ni, Yuefei Jin, Shuaiyin Chen, Guangcai Duan, and Rongguang Zhang. "Application of CRISPR/Cas Systems in the Nucleic Acid Detection of Infectious Diseases." Diagnostics 12, no. 10 (October 11, 2022): 2455. http://dx.doi.org/10.3390/diagnostics12102455.

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The CRISPR/Cas system is a protective adaptive immune system against attacks from foreign mobile genetic elements. Since the discovery of the excellent target-specific sequence recognition ability of the CRISPR/Cas system, the CRISPR/Cas system has shown excellent performance in the development of pathogen nucleic-acid-detection technology. In combination with various biosensing technologies, researchers have made many rapid, convenient, and feasible innovations in pathogen nucleic-acid-detection technology. With an in-depth understanding and development of the CRISPR/Cas system, it is no longer limited to CRISPR/Cas9, CRISPR/Cas12, and other systems that had been widely used in the past; other CRISPR/Cas families are designed for nucleic acid detection. We summarized the application of CRISPR/Cas-related technology in infectious-disease detection and its development in SARS-CoV-2 detection.
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Yang, Hui, and Dinshaw J. Patel. "New CRISPR-Cas systems discovered." Cell Research 27, no. 3 (February 21, 2017): 313–14. http://dx.doi.org/10.1038/cr.2017.21.

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Sternberg, Samuel H., Hagen Richter, Emmanuelle Charpentier, and Udi Qimron. "Adaptation in CRISPR-Cas Systems." Molecular Cell 61, no. 6 (March 2016): 797–808. http://dx.doi.org/10.1016/j.molcel.2016.01.030.

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Dissertations / Theses on the topic "CRISPR-Cas Systems"

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Bernheim, Aude. "The distribution of CRISPR-Cas systems is affected by interactions with DNA repair pathways." Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCB070/document.

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Les systèmes CRISPR-Cas confèrent aux bactéries une immunité adaptative contre les éléments génétiques mobiles jouant ainsi un rôle important dans l’évolution bactérienne. Cependant, moins de la moitié des génomes bactériens encodent des systèmes CRISPR-Cas ; cela, malgré la protection qu’ils confèrent et leur haut taux de transfert horizontal. Des hypothèses telles que le coût des phénomènes d’auto-immunité ou de posséder des défenses adaptatives plutôt qu’innées ont été mises en avant pour expliquer ce paradoxe. Je propose une nouvelle hypothèse complémentaire : le contexte génétique jouerait un rôle important dans la fixation d’un système CRISPR-Cas après son transfert. Plus précisément, j’ai étudié comment les interactions entre les systèmes de réparation de l’ADN et les CRISPR-Cas influencent la distribution de ces derniers. Pour cela, j’ai d’abord examiné finement la distribution des systèmes CRISPR-Cas dans les génomes bactériens. J’ai ensuite analysé les co-occurences des systèmes de réparation de l’ADN et des CRISPR-Cas et démontré l’existence d’associations positives et négatives entre eux. Enfin, je me suis concentrée sur une des associations négatives découvertes pour valider mes prédictions expérimentalement et comprendre les mécanismes moléculaires sous-jacents. Mes travaux permettent de mieux comprendre les interactions complexes entre systèmes de réparation de l’ADN et CRISPR-Cas et démontrent la nécessite d’accommodation des CRISPR-Cas à un contexte génétique pour être sélectionnés et maintenus dans les génomes bactériens
CRISPR-Cas systems confer bacteria and archea an adaptative immunity against phages and other invading genetic elements playing an important role in bacterial evolution. Only 47% of bacterial genomes harbor a CRISPR-Cas system despite their high rate of horizontal transfer. Hypothesis such as the cost of autoimmu- nity or the trade off between a constitutive or an inducible defense system have been put forward to explain this paradox. I propose that the genetic background plays an important role in the process of maintaining a CRISPR-Cas system af- ter its transfer. More precisely I hypothesized that CRISPR-Cas systems interact with DNA repair pathways. To test this idea, we detected DNA repair pathways and CRISPR-Cas systems in bacterial genomes and studied their co-occurences. We report both positive and negative associations that we interpret as poten- tial antagonistic or synergistic interactions. We then focused on one interaction to validate our result experimentally and explored molecular mechanisms behind those interactions. My findings give insights on the complex interactions between CRISPR-Cas systems and DNA repair mechanisms in bacteria and provide a first example on the necessity of accommodation of CRISPR-Cas systems to a specific genetic context to be selected and maintained in bacterial genomes
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Vyhovskyi, Danylo. "In vivo studies of CRISPR adaptation mechanism and specificity." Electronic Thesis or Diss., Sorbonne université, 2023. http://www.theses.fr/2023SORUS729.

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Cette thèse examine les mécanismes de l'immunité adaptative CRISPR-Cas chez les procaryotes, en utilisant principalement le système de type I-E chez Escherichia coli, en se concentrant sur le processus d'acquisition de spacers et la spécificité du système. Elle éclaire la dynamique de génération de spacers et leur intégration dans les CRISPR-arrays, en comparant les modes d'adaptation naïve et primed. L'étude révèle qu'une séquence proximale à un PAM (protospacer adjacent motif) particulier entrave l'acquisition de spacers en mode primed, fournissant un identifiant distinct pour les spacers acquis naturellement. L'étude révèle en outre le rôle des enzymes non-Cas, liées aux voies de réparation de l'ADN, dans la génération et le traitement des spacers, contribuant à l'adaptation et à l'interférence du CRISPR.Un autre volet de l'étude identifie les dangers potentiels posés par les effets hors cible causés par l'enzyme guidée par l'ARN Cas9 (dCas9) qui peut inadvertamment réduire au silence les gènes. Cela se produit lorsqu'il y a une correspondance minimale de seulement quatre nucléotides entre l'ARNg et la cible à l'intérieur de la séquence proximale du PAM, soulignant la nécessité d'une conception expérimentale soigneuse dans la recherche CRISPR-Cas. Dans l'ensemble, la thèse élargit la compréhension des mécanismes moléculaires complexes derrière l'adaptation du CRISPR, soulignant le rôle des protéines non-Cas et l'importance d'un contexte génétique spécifique des séquences proximales à un PAM, conduisant au développement d'outils de génie génétique plus précis et efficaces
This thesis investigates the mechanisms of the CRISPR-Cas adaptive immunity in prokaryotes, primarily using the type I-E system in Escherichia coli, focusing on the spacer acquisition process and the system's specificity. It sheds light on the dynamics of spacers generation and integration into CRISPR arrays, comparing naive and primed adaptation modes. The study reveals that a particular PAM (protospacer adjacent motif) - proximal sequence impedes spacer acquisition in the primed mode, providing a distinct identifier for naturally acquired spacers. The study further reveals the role of non-Cas enzymes, linked to DNA repair pathways, in spacer generation and processing, contributing to CRISPR adaptation and interference.Another course of the study identifies potential hazards posed by off-target effects caused by Cas9 (dCas9) RNA-guided enzyme that can inadvertently silence genes. This occurs when there's a minimal match of just four nucleotides between the gRNA and the target within the PAM-proximal sequence, emphasizing the need for careful experimental design in CRISPR-Cas research.Overall, the thesis expands understanding of the complex molecular mechanisms behind CRISPR adaptation, highlighting the role of non-Cas proteins and the significance of a specific genetic context of seed sequences, leading to the development of more precise and efficient genetic engineering tools
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Alkhnbashi, Omer S. [Verfasser], and Rolf [Akademischer Betreuer] Backofen. "Computational characterisation of genomic CRISPR-Cas systems in archaea and bacteria." Freiburg : Universität, 2017. http://d-nb.info/1139210904/34.

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Ansai, Satoshi. "Targeted mutagenesis in medaka using targetable nuclease systems." Kyoto University, 2016. http://hdl.handle.net/2433/215591.

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Kyoto University (京都大学)
0048
新制・課程博士
博士(農学)
甲第19765号
農博第2161号
新制||農||1039(附属図書館)
学位論文||H28||N4981(農学部図書室)
32801
京都大学大学院農学研究科応用生物科学専攻
(主査)教授 佐藤 健司, 教授 澤山 茂樹, 准教授 田川 正朋
学位規則第4条第1項該当
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Zhu, Houxiang. "Optimal gRNA design of different CRISPR-Cas systems for DNA and RNA editing." Miami University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=miami1556307865151938.

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Brendel, Jutta [Verfasser]. "Charakterisierung der Prozessierungs-und Interferenzaktivität des CRISPR/Cas-Systems in Haloferax volcanii / Jutta Brendel." Ulm : Universität Ulm. Fakultät für Naturwissenschaften, 2014. http://d-nb.info/1053705611/34.

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Hille, Frank [Verfasser]. "Investigation of Spacer Acquisition Mechanisms in Type V-A CRISPR-Cas Systems / Frank Hille." Berlin : Humboldt-Universität zu Berlin, 2020. http://d-nb.info/1215570341/34.

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Staub, Dillon. "Bio-Inspired Hardware Security Defenses: A CRISPR-Cas-Based Approach for Detecting Trojans in FPGA Systems." University of Cincinnati / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1563872470616901.

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Silva, Caroline Caetano da. "O nr2e1 influencia o comportamento exploratório, mas não é necessário para a diferenciação hormonal hipofisária no zebrafish (Danio rerio)." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5135/tde-16082017-133351/.

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Hipopituitarismo congênito é caracterizado por deficiência hormonal múltipla devido a mutações de fatores de transcrição envolvidos na embriogênese hipofisária. As células-tronco estão presentes na hipófise e são caracterizadas por dar origem a uma célula progenitora e uma célula indiferenciada por divisão assimétrica. Estão envolvidas na hipófise em processos de alta demanda metabólica em diferentes fases da vida. Em estudos prévios, observou-se o acúmulo dos marcadores de células-tronco Sox2 e Nr2e1 no camundongo Ames, que apresenta mutação no gene Prop1. O Sox2 é o marcador consenso de células-tronco na hipófise enquanto que o Nr2e1, nunca antes caracterizado na hipófise, é essencial para a manutenção de células-tronco e neogenese no cérebro. A perda de função deste gene pode causar agressão e falta de instinto materno em camundongos. Com isso, o objetivo desse projeto foi utilizar o animal modelo zebrafish para avaliar o papel repressor do gene prop1 e caracterizar o gene nr2e1 bem como, confirmar se o mesmo está envolvido com a diferenciação terminal na hipófise, e sua interferência no comportamento do animal mutado. O zebrafish se encaixa adequadamente nesse projeto pois é de fácil manutenção, econômico e com rápido desenvolvimento. No presente estudo criou-se 2 modelos de zebrafish utilizando-se a técnica de edição genômica conhecida como CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) para nocautear os genes prop1 e nr2e1. Esta técnica permite uma interrupção específica e substituição de bases no genoma, resultando em uma alta especificidade, baixa toxicidade celular e é herdável. O zebrafish homozigoto com mutação no gene nr2e1 se desenvolve e reproduz como o animal controle, porém apresenta um comportamento mais exploratório quando comparado com o animal selvagem e o heterozigoto. A imunofluorescência para o anticorpo Sox2 no animal mutado mostrou se diferente do selvagem, pois apresenta um aumento da expressão temporal e o mesmo não se colocaliza com o Nr2e1. A imunofluorescência feita com os hormônios não se mostrou diferente entre o mutado e o selvagem. Conclui-se diante dos achados de normalidade do desenvolvimento, fertilidade, ausência de co-localização com o gene Sox2 e presença de hormônios como Tsh, Fsh e Gh, que o gene nr2e1 não é crucial na diferenciação terminal na hipófise porém o animal mutado apresenta um comportamento diferente do animal selvagem. Os resultados da caracterização do zebrafish com mutação no gene prop1 ainda estão em andamento devido a dificuldade de se estabelecer essa linhagem
Congenital hypopituitarism is characterized by multiple hormone deficiencies due to mutations in transcription factors involved in pituitary embryogenesis. Stem cells, which by definition can each give rise to a progenitor and an undifferentiated cell by asymmetric division, are present in the pituitary gland and are important during periods of high metabolic demand in different phases of life. In previous studies, the accumulation of the stem cell markers Sox2 and Nr2e1 was observed in the Ames mouse, which harbors a mutation in Prop1. Sox2 is the consensus stem cell marker in the pituitary gland, while the role of Nr2e1 in the pituitary development has not been characterized although it is essential for neural stem cell maintenance and neogenesis in the brain and its loss of function causes pathological aggression and lack of maternal instinct in mice. In this project, the zebrafish animal model was used to characterize the role of nr2e1, to confirm whether this gene can be involved in the pituitary terminal differentiation, and to determine the effects of this gene on animal behavior. The zebrafish is a particularly appropriate model for use in this project because it is easy to maintain, is economical, and has a rapid metabolism and growth rate. In the present study, we created 2 zebrafish models by knocking out prop1 and nr2e1 using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) genome-editing technique. This technique enables highly specific gene/reading frame interruption and/or base substitution in the genome, with low cellular toxicity and high heritability. Zebrafish with homozygous nr2e1 mutations develop and reproduce similarly to wild-type zebrafish, but present a more exploratory behavioral pattern compared to wild-type and heterozygous zebrafish. Based on immunofluorescence, Sox2 expression was higher in the mutant zebrafish than in the wild type and was not co-localized with Nr2e1 expression. Hormone expression did not differ between wild-type and mutant zebrafish. We conclude that nr2e1 is not crucial in the terminal differentiation of the hormone-forming pituitary gland; however, it induces a distinct behavioral phenotype at the larval stage. Analyses of zebrafish harboring a prop1 mutation are ongoing owing to issues with the establishment of the lineage
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Zhu, Jiang. "PART I CRYSTAL STRUCTURE OF A DIMERIZATION DOMAIN OF DROSOPHILA CAPRIN. PART II CHARACTERIZATION OF TWO CAS13B CRISPR-CAS SYSTEMS FROM PORPHYROMONAS GINGIVALIS." OpenSIUC, 2018. https://opensiuc.lib.siu.edu/dissertations/1503.

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PART I: CRYSTAL STRUCTURE OF A DIMERIZATIO DOMAIN OF DROSOPHILA CAPRIN Drosophila Melanogaster Caprin (dCaprin) shares conversed HR1 domain with Caprin protein family members, which are RNA binding proteins that play critical roles in many important biological processes, such as synaptic plasticity, stress response, innate immune response and cellular proliferation. One of the Caprin protein family members, Caprin-1, is involved in the pathway of several human diseases, including breast cancer, neurodegenerative disorders, osteosarcoma, hearing loss, and viral infection. The functions of Caprin protein relies on their molecular interactions. Several direct interactions have been established between Caprin-1 and the Fragile X mental retardation protein (FMRP), Ras-GAP SH3 domain-binding protein 1 (G3BP1), and the Japanese encephalitis virus (JEV) core protein. We have determined the crystal structures of a fragment (residues 187-309) of Drosophila Melanogaster Caprin (dCaprin), which mediates homodimerization through a substantial interface created by a mainly alpha-helical fold. A larger hollow surface is created by homodimerization suggesting a protein binding groove. The FMRP binding should not affect dCaprin homodimerization for an integral alpha-helix in the dimeric dCaprein which formed by the FMRP interacting sequence motif. PART II: CHARACTERIZATION OF TWO TYPE VI-B CRESPR SYSTEMS: PGI5CAS1B AND PGI8CAS13B WHICH EFFECTOR PROTEINS ARE CAPABLE OF PROCESSING PRE-CRRNA INTO MATURE CRRNA CRISPR-Cas adaptive immune system protects microorganism from foreign nucleic acids invasion through endonucleases activity guided by RNA, which system has turned to a powerful genome editing tool applied to a multifold species, ranging from bacteria to human. Pgi5Cas13b and Pgi8Cas13b are identified by a computational sequence database mining approach, the CRISPR arrays lack of Cas1 and Cas2 encoding genes but contain a large candidate effector protein around 1,200 amino acids. They can be potentially classified as subtype VI-B CRISPR-Cas systems. We characterized the mature crRNA for Pgi5Cas13b and Pgi8Cas13b via Northern blot and small RNA sequencing. By EMSA (Electrophoretic mobility shift assay) experiments, we identified the binding constant between Pgi5Cas1b/Pgi8Cas13b and their corresponding crRNAs. The CRISPR loci of two of the Cas13b systems were cloned in pACYC vector and expressed in E. coli cells. Small RNAs were extracted and characterized by Northern Blotting and NGS (small RNA-seq) methods. The NGS results revealed the exact sequences of the crRNAs, which show a few new features not previously observed in other systems, including the longest spacer-derived sequences (32 and 31 nt), spacer-derived sequences flanking both ends of the full DR-derived sequence. The results also indicate different rules of pre-crRNA processing by the Pgi5Cas13b and Pgi8Cas13b systems. The characterization of these CRISPR systems extends the application of CRISPR based genome editing tools and promotes the development of single transcript manipulation tools.
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Books on the topic "CRISPR-Cas Systems"

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Barrangou, Rodolphe, and John van der Oost, eds. CRISPR-Cas Systems. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-662-45794-8.

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Barrangou, Rodolphe, and John van der Oost, eds. CRISPR-Cas Systems. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/978-3-642-34657-6.

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Oost, John van der, and Rodolphe Barrangou. CRISPR-Cas systems: RNA-mediated adaptive immunity in bacteria and archaea. Heidelberg: Springer, 2013.

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Saunders, Sita Johanna. CRISPRmap: An automated classification of repeat conservation in prokaryotic adaptive immune systems. Freiburg: Universität, 2013.

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Halpin-Healy, Tyler Sheehan. Structure and Function of a Transposon-Encoded CRISPR-Cas System. [New York, N.Y.?]: [publisher not identified], 2021.

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Doudna, Jennifer A., and Prashant Mali. CRISPR-Cas. Cold Spring Harbor Laboratory Press, 2016.

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Kamruzzaman, Muhammad, Graciela Castro Escarpulli, and Aixin Yan, eds. CRISPR-Cas Systems in Bacteria and Archaea. Frontiers Media SA, 2022. http://dx.doi.org/10.3389/978-2-88974-963-8.

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Oost, John van der, and Rodolphe Barrangou. CRISPR-Cas Systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea. Springer, 2012.

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Oost, John van der, and Rodolphe Barrangou. CRISPR-Cas Systems: RNA-mediated Adaptive Immunity in Bacteria and Archaea. Springer, 2015.

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Technikfolgenabschätzung des CRISPR/Cas-Systems: Über Die Anwendung in der Menschlichen Keimbahn. De Gruyter, Inc., 2019.

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Book chapters on the topic "CRISPR-Cas Systems"

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Mojica, Francisco J. M., and Roger A. Garrett. "Discovery and Seminal Developments in the CRISPR Field." In CRISPR-Cas Systems, 1–31. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_1.

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Amitai, Gil, and Rotem Sorek. "Roles of CRISPR in Regulation of Physiological Processes." In CRISPR-Cas Systems, 251–66. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_10.

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Horvath, Philippe, Giedrius Gasiunas, Virginijus Siksnys, and Rodolphe Barrangou. "Applications of the Versatile CRISPR-Cas Systems." In CRISPR-Cas Systems, 267–86. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_11.

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Banfield, Jillian F. "CRISPRs in the Microbial Community Context." In CRISPR-Cas Systems, 287–91. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_12.

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Pourcel, Christine, and Christine Drevet. "Occurrence, Diversity of CRISPR-Cas Systems and Genotyping Implications." In CRISPR-Cas Systems, 33–59. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_2.

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Makarova, Kira S., and Eugene V. Koonin. "Evolution and Classification of CRISPR-Cas Systems and Cas Protein Families." In CRISPR-Cas Systems, 61–91. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_3.

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Arslan, Zihni, Edze R. Westra, Rolf Wagner, and Ümit Pul. "Regulation of CRISPR-Based Immune Responses." In CRISPR-Cas Systems, 93–113. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_4.

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Charpentier, Emmanuelle, John van der Oost, and Malcolm F. White. "crRNA Biogenesis." In CRISPR-Cas Systems, 115–44. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_5.

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Staals, Raymond H. J., and Stan J. J. Brouns. "Distribution and Mechanism of the Type I CRISPR-Cas Systems." In CRISPR-Cas Systems, 145–69. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_6.

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Dupuis, Marie-Ève, and Sylvain Moineau. "Type II: Streptococcus thermophilus." In CRISPR-Cas Systems, 171–200. Berlin, Heidelberg: Springer Berlin Heidelberg, 2012. http://dx.doi.org/10.1007/978-3-642-34657-6_7.

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Conference papers on the topic "CRISPR-Cas Systems"

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Sakr, N. "CONTROL OF GENE EXPRESSION BY CRISPR-CAS SYSTEMS." In Конференция «Перспективы применения генной терапии и биомедицинского клеточного продукта» с блоком летней школы для молодых ученых. Федеральное государственное бюджетное учреждение «Национальный медицинский исследовательский центр эндокринологии» Министерства здравоохранения Российской Федерации, 2022. http://dx.doi.org/10.14341/gnct-2022-50.

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Potyseva, A. S., A. N. Arseniev, P. A. Selkova, A. A. Vasilieva, A. S. Melnikov, P. Yu Serdobintsev, and M. A. Khodorkovskii. "COMPARISON OF COLLATERAL ACTIVITY OF CRISPR CS12A ORTHOLOGS FOR THE DEVELOPMENT OF NOVEL DIAGNOSTIC SYSTEMS." In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-361.

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CRISPR/Cas systems are defense systems for bacteria and archaea. Several classes of these systems have now been characterized. In addition to the main directed nuclease activity, type II class V systems possess specific collateral activity, which formed the basis for a new type of diagnostic systems. In the result of this work we obtained data characterizing the collateral activity of two orthologues of this class and demonstrated that the CRISPR system ScCas12a has greater specificity compared to AsCas12a.
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Tyumentseva, M. A., A. I. Tyumentsev, and V. G. Akimkin. "DEVELOPMENT OF APPROACHES FOR DETECTION OF GENOME-INTEGRATED PROVIRAL DNA OF THE HUMAN IMMUNODEFICIENCY VIRUS (HIV-1) IN ULTRA LOW CONCENTRATIONS USING THE CRISPR/CAS SYSTEM." In Molecular Diagnostics and Biosafety. Federal Budget Institute of Science 'Central Research Institute for Epidemiology', 2020. http://dx.doi.org/10.36233/978-5-9900432-9-9-118.

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For the effective functioning of supervisory and health monitoring services, it is necessary to introduce modern molecular technologies into their practice. Therefore, the task of developing new effective methods for detecting pathogen, for example HIV, based on CRISPR/CAS genome editing systems, remains urgent. In the present work, guide RNAs and specific oligonucleotides were developed for preliminary amplification of highly conserved regions of the HIV-1 genome. The developed guide RNAs make it possible to detect single copies of HIV-1 proviral DNA in vitro as part of CRISPR/CAS ribonucleoprotein complexes in biological samples after preliminary amplification.
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Pernak, Elizaveta, Mariia Vladimirova, Viktoria Muntyan, Alexey Afonin, and Marina Roumiantseva. "ANALYSIS OF CRISPR CASSETTE ELEMENTS IN NATIVE ISOLATES OF SINORHIZOBIUM MELILOTI ISOLATED IN THE CENTRAL ASIAN ORIGIN OF PLANT DIVERSITY." In 23rd SGEM International Multidisciplinary Scientific GeoConference 2023. STEF92 Technology, 2023. http://dx.doi.org/10.5593/sgem2023/6.1/s25.13.

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CRISPR-Cas adaptive immunity systems of prokaryotes have been identified in 42% of bacteria and 85% of archaea [1] and are actively used in genetic engineering. However, in nitrogen-fixing nodule bacteria required for agriculture, these systems are very poorly characterized. Therefore, in this study, we evaluated the diversity of CRISPR-Cas systems in the genomes of 39 isolates of alfalfa nodule bacteria Sinorhizobium meliloti recovered from soil samples from Kazakhstan and Turkmenistan, which belong to the Central Asian origion of plants diversity. The diversity of detected CRISPR elements was assessed by changes in the size of the PCR amplified DNA fragments. As a result, the presence of 7 species-specific CRISPR loci (CR1- CR7) was shown for each of 39 isolates, of which three (CR4, CR5 and CR6) were selected for further analysis. It was shown that structural changes in the analyzed CRISPR loci sequences were significantly 2-fold more frequent in strains from Turkmenistan than in strains from Kazakhstan (?2=4.07, P less than 0.05). The most frequent changes in the size of the amplified sequences were detected at the CR6 locus (frequency 0.39), and 7.5-fold less frequent at the CR4 locus. Analysis of the nucleotide sequences of the loci for which changes in the size of amplified fragments were detected showed that at the CR4 locus one spacer was replaced with another; at the CR6 locus a deletion of the spacer and one direct repeat were involved. No changes were detected in the sequences of the CR-5 locus. The results suggest that strains in which changes in cassette compositions have occurred may have significant differences in resistance to lytic phages. The possibility of directionally modulating the spacers sequence in the CRISPR loci of nodule bacteria used in bioproducts may undoubtedly be of interest for agrobiotechnology.
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Yu, Eun-Sil, Byoung-Hoon Kang, Hamin Na, and Ki-Hun Jeong. "Nanoplasmonic isothermal PCR assay with CRISPR/Cas for real-time SARS-CoV-2 detection." In MOEMS and Miniaturized Systems XXII, edited by Wibool Piyawattanametha, Yong-Hwa Park, and Hans Zappe. SPIE, 2023. http://dx.doi.org/10.1117/12.2650879.

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Kapitonova, M. A., A. V. Shabalina, V. G. Dedkov, and A. S. Dolgova. "DEVELOPMENT OF CRISPR-CAS12A BASED DIAGNOSTIC SYSTEM FOR GUANARITO VIRUS DETECTION COMBINED WITH ISOTHERMAL AMPLIFICATION IN ONE POT." In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-327.

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CRISPR-Cas based diagnostic systems become more popular due to adaptability, simplicity and rapidness of analyses. In particular, the prospect of combining it with an isothermal amplification opens up new possibilities. In this work, we develop a detection system of the Guanarito virus based on Cas12a with RPA amplification in one pot. After several parameters optimization the analytical sensitivity is evaluated as 102 copies/µl.
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"Bioinformatics analysis of the structures of CRISPR/Cas-systems in the genomes of phytopathogenic bacteria." In Plant Genetics, Genomics, Bioinformatics, and Biotechnology. Institute of Cytology and Genetics, Siberian Branch of the Russian Academy of Sciences, 2019. http://dx.doi.org/10.18699/plantgen2019-139.

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Zhang, Liuyijia. "CRISPR/Cas system in human genetic diseases." In Third International Conference on Biological Engineering and Medical Science (ICBioMed2023), edited by Alan Wang. SPIE, 2024. http://dx.doi.org/10.1117/12.3012830.

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Muntyan, Victoria S., Alla S. Saksaganskaia, Alexey N. Muntyan, Mariia E. Vladimirova, and Marina L. Roumiantseva. "STRESS AND IMMUNITY OF NODULE BACTERIA SINORHIZOBIUM MELILOTI: LOCALIZATION, POLYMORPHISM AND PHYLOGENY OF GENETIC DETERMINANTS." In 22nd SGEM International Multidisciplinary Scientific GeoConference 2022. STEF92 Technology, 2022. http://dx.doi.org/10.5593/sgem2022/6.1/s25.15.

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Sinorhizobium meliloti are agriculturally valuable species of soil bacteria that form nitrogen-fixing symbiosis with alfalfa plants. Global climate changes lead to an increase of agricultural areas subjected to salinity. Current knowledge about about high-salt stress impact on soil saprophitic root nodulated microsymbionts of legumes is weakly studied and rhizobia gene pool responsible for salt tolerance are fragment and far from clear. An increase of bacteria nonspecific resistance (immune status) to unfavorable stress factors can occur through the induction of defense mechanisms like restrictionmodification systems and CRISPR/cas systems which are aimed to protect bacteria cells from bacteriophages widespread in soil microbiome. The aim of this research was to evaluate the role of the megaplasmid pSymA in the formation of ecological genome of S. meliloti, which is related to stress tolerance and to determine the location of elements of adaptive immune systems protecting root nodule bacteria against external foreign DNA. The analysis was done on 11 genes, products of which involved in response to ion stress and synthesis of osmoprotectors. It was found that 6 out of 11 genes were found in the genomes of all analyzed S. meliloti strains, while it was not a case for other 5 genes. It was found that, unlike chromosome, megaplasmid I of S. meliloti accumulated copies of 4 from 5 genes, except kdpA gene, which is represented by a single copy and localized on megaplasmid I in all so far studied strains. It was predicted that closest phylogenetic relatives of genes whose products are involved in response to ion stress as well in synthesis of osmoprotectors are homologous genes of closely related S. medicae species. The exception was for betI2, for which the closest phylogenetic relative was homologous gene of Klebsiella pneumonia, and another exception is kdpA gene introduced onto megaplasmid-I from actinobacteria. Regarding elements of immune systems it was revealed that nonsymbiotic plasmids of S. meliloti harbored incomplete elements of RMS-I, -II, and - III systems, while the 4 complete RMS-IV systems were detected on a single plasmid. It was found out that corresponding methylases had similarities with similar enzymes detected in nitrogen-fixing strains of Agrobacterium tumefaciens, Mezorhizobium sp., Bradyrhizobium sp. CRISPR sequences were not detected on megaplasmid-I, while they were on chromosome, megaplasmid-II and on cryptic plasmids. So, it was concluded that megaplasmid-I of S. meliloti are enriched in copies of genes related to osmotic stress tolerance, but it role in immune status of rhizobia is requested further elucidation.
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Zhang, Hanlin. "Therapeutic applications of CRISPR-Cas system in infectious diseases." In Third International Conference on Biological Engineering and Medical Science (ICBioMed2023), edited by Alan Wang. SPIE, 2024. http://dx.doi.org/10.1117/12.3012858.

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Reports on the topic "CRISPR-Cas Systems"

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Sanford, Jack, and John Weldon. The Biology of Native and Adapted CRISPR-Cas Systems. Journal of Young Investigators, November 2018. http://dx.doi.org/10.22186/jyi.35.5.81-91.

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Bagley, Margo. Genome Editing in Latin America: CRISPR Patent and Licensing Policy. Inter-American Development Bank, July 2021. http://dx.doi.org/10.18235/0003409.

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The power and promise of genome editing, CRISPR specifically, was first realized with the discovery of CRISPR loci in the 1980s.i Since that time, CRISPR-Cas systems have been further developed enabling genome editing in virtually all organisms across the tree of life.i In the last few years, we have seen the development of a diverse set of CRISPR-based technologies that has revolutionized genome manipulation.ii Enabling a more diverse set of actors than has been seen with other emerging technologies to redefine research and development for biotechnology products encompassing food, agriculture, and medicine.ii Currently, the CRISPR community encompasses over 40,000 authors at 20,000 institutions that have documented their research in over 20,000 published and peer-reviewed studies.iii These CRISPR-based genome editing tools have promised tremendous opportunities in agriculture for the breeding of crops and livestock across the food supply chain. Potentially addressing issues associated with a growing global population, sustainability concerns, and possibly help address the effects of climate change.i These promises however, come along-side concerns of environmental and socio-economic risks associated with CRISPR-based genome editing, and concerns that governance systems are not keeping pace with the technological development and are ill-equipped, or not well suited, to evaluate these risks. The Inter-American Development Bank (IDB) launched an initiative in 2020 to understand the complexities of these new tools, their potential impacts on the LAC region, and how IDB may best invest in its potential adoption and governance strategies. This first series of discussion documents: “Genome Editing in Latin America: Regulatory Overview,” and “CRISPR Patent and Licensing Policy” are part of this larger initiative to examine the regulatory and institutional frameworks surrounding gene editing via CRISPR-based technologies in the Latin America and Caribbean (LAC) regions. Focusing on Argentina, Bolivia, Brazil, Colombia, Honduras, Mexico, Paraguay, Peru, and Uruguay, they set the stage for a deeper analysis of the issues they present which will be studied over the course of the next year through expert solicitations in the region, the development of a series of crop-specific case studies, and a final comprehensive regional analysis of the issues discovered.
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Kuiken, Todd, and Jennifer Kuzma. Genome Editing in Latin America: Regional Regulatory Overview. Inter-American Development Bank, July 2021. http://dx.doi.org/10.18235/0003410.

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The power and promise of genome editing, CRISPR specifically, was first realized with the discovery of CRISPR loci in the 1980s.3 Since that time, CRISPR-Cas systems have been further developed enabling genome editing in virtually all organisms across the tree of life.3 In the last few years, we have seen the development of a diverse set of CRISPR-based technologies that has revolutionized genome manipulation.4 Enabling a more diverse set of actors than has been seen with other emerging technologies to redefine research and development for biotechnology products encompassing food, agriculture, and medicine.4 Currently, the CRISPR community encompasses over 40,000 authors at 20,000 institutions that have documented their research in over 20,000 published and peer-reviewed studies.5 These CRISPR-based genome editing tools have promised tremendous opportunities in agriculture for the breeding of crops and livestock across the food supply chain. Potentially addressing issues associated with a growing global population, sustainability concerns, and possibly help address the effects of climate change.4 These promises however, come along-side concerns of environmental and socio-economic risks associated with CRISPR-based genome editing, and concerns that governance systems are not keeping pace with the technological development and are ill-equipped, or not well suited, to evaluate these risks. The Inter-American Development Bank (IDB) launched an initiative in 2020 to understand the complexities of these new tools, their potential impacts on the LAC region, and how IDB may best invest in its potential adoption and governance strategies. This first series of discussion documents: “Genome Editing in Latin America: Regulatory Overview,” and “CRISPR Patent and Licensing Policy” are part of this larger initiative to examine the regulatory and institutional frameworks surrounding gene editing via CRISPR-based technologies in the Latin America and Caribbean (LAC) regions. Focusing on Argentina, Bolivia, Brazil, Colombia, Honduras, Mexico, Paraguay, Peru, and Uruguay, they set the stage for a deeper analysis of the issues they present which will be studied over the course of the next year through expert solicitations in the region, the development of a series of crop-specific case studies, and a final comprehensive regional analysis of the issues discovered.
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Zarate, Sebastian, Ilaria Cimadori, Maria Mercedes Roca, Michael S. Jones, and Katie Barnhill-Dilling. Assessment of the Regulatory and Institutional Framework for Agricultural Gene Editing via CRISPR-based Technologies in Latin America and the Caribbean. Inter-American Development Bank, May 2023. http://dx.doi.org/10.18235/0004904.

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Genome editing tools have promised tremendous opportunities in agriculture for breeding crops and livestock across the food supply chain. Potentially addressing issues associated with a growing global population, sustainability concerns, and possibly helping address the effects of climate change (Kuiken, Barrangou, and Grieger 2021). These promises come alongside environmental, cultural, and socio-economic risks. Including concerns that governance systems are not keeping pace with technological developments and are ill-equipped, or not well suited to evaluate risks new genome editing tools may introduce. Understanding these complex, dynamic interactions across the LAC region is important to inform appropriate and acceptable regional governance and investment strategies. The power and promise of gene editing, CRISPR specifically, were first realized with the discovery of CRISPR loci in the 1980s (Anzalone, Koblan, and Liu 2020). Since that time, CRISPR-Cas systems have been further developed enabling genome editing in virtually all organisms across the tree of life (Anzalone, Koblan, and Liu 2020). Gene editing is not a singular technology or technique; it refers most often to a set of techniques that enable the manipulation of a genome with greater precision than previous iterations of genetic engineering (Shukla-Jones, Friedrichs, and Winickoff 2018b). The Inter-American Development Bank partnered with North Carolina State Universitys Genetic Engineering and Society (GES) Center to assess the regulatory and institutional frameworks surrounding gene-editing via CRISPR-based technologies in the Latin America and Caribbean (LAC) regions. The project studied the following core components: Current Policy Evaluation: Understanding what the future may hold requires a critical examination of the current status of the regulatory landscape. Analysis of the existing regulatory systems for agricultural biotechnologies throughout Latin America and how they included considerations for novel biotechnology strategies such as gene editing through CRISPR technologies were done. Forecasting and Future Policy Scenario Analysis: Potential products created through gene editing may face very different situations on the ground, depending on countries diverse regulations and market structures. To clarify the potential impacts of regulatory reforms, we included concrete case studies in our analysis. Identifying investment priorities: The diversity of the region naturally means that countries will have unique priorities and needs with respect to investment in agricultural biotechnology development and regulatory infrastructure. The document evaluates the accomplishments of the region in the development of gene edited products, highlighting both private and public sector innovations.
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Burns, Malcom, and Gavin Nixon. Literature review on analytical methods for the detection of precision bred products. Food Standards Agency, September 2023. http://dx.doi.org/10.46756/sci.fsa.ney927.

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The Genetic Technology (Precision Breeding) Act (England) aims to develop a science-based process for the regulation and authorisation of precision bred organisms (PBOs). PBOs are created by genetic technologies but exhibit changes which could have occurred through traditional processes. This current review, commissioned by the Food Standards Agency (FSA), aims to clarify existing terminologies, explore viable methods for the detection, identification, and quantification of products of precision breeding techniques, address and identify potential solutions to the analytical challenges presented, and provide recommendations for working towards an infrastructure to support detection of precision bred products in the future. The review includes a summary of the terminology in relation to analytical approaches for detection of precision bred products. A harmonised set of terminology contributes towards promoting further understanding of the common terms used in genome editing. A review of the current state of the art of potential methods for the detection, identification and quantification of precision bred products in the UK, has been provided. Parallels are drawn with the evolution of synergistic analytical approaches for the detection of Genetically Modified Organisms (GMOs), where molecular biology techniques are used to detect DNA sequence changes in an organism’s genome. The scope and limitations of targeted and untargeted methods are summarised. Current scientific opinion supports that modern molecular biology techniques (i.e., quantitative real-time Polymerase Chain Reaction (qPCR), digital PCR (dPCR) and Next Generation Sequencing (NGS)) have the technical capability to detect small alterations in an organism’s genome, given specific prerequisites of a priori information on the DNA sequence of interest and of the associated flanking regions. These techniques also provide the best infra-structure for developing potential approaches for detection of PBOs. Should sufficient information be known regarding a sequence alteration and confidence can be attributed to this being specific to a PBO line, then detection, identification and quantification can potentially be achieved. Genome editing and new mutagenesis techniques are umbrella terms, incorporating a plethora of approaches with diverse modes of action and resultant mutational changes. Generalisations regarding techniques and methods for detection for all PBO products are not appropriate, and each genome edited product may have to be assessed on a case-by-case basis. The application of modern molecular biology techniques, in isolation and by targeting just a single alteration, are unlikely to provide unequivocal evidence to the source of that variation, be that as a result of precision breeding or as a result of traditional processes. In specific instances, detection and identification may be technically possible, if enough additional information is available in order to prove that a DNA sequence or sequences are unique to a specific genome edited line (e.g., following certain types of Site-Directed Nucelase-3 (SDN-3) based approaches). The scope, gaps, and limitations associated with traceability of PBO products were examined, to identify current and future challenges. Alongside these, recommendations were made to provide the infrastructure for working towards a toolkit for the design, development and implementation of analytical methods for detection of PBO products. Recognition is given that fully effective methods for PBO detection have yet to be realised, so these recommendations have been made as a tool for progressing the current state-of-the-art for research into such methods. Recommendations for the following five main challenges were identified. Firstly, PBOs submitted for authorisation should be assessed on a case-by-case basis in terms of the extent, type and number of genetic changes, to make an informed decision on the likelihood of a molecular biology method being developed for unequivocal identification of that specific PBO. The second recommendation is that a specialist review be conducted, potentially informed by UK and EU governmental departments, to monitor those PBOs destined for the authorisation process, and actively assess the extent of the genetic variability and mutations, to make an informed decision on the type and complexity of detection methods that need to be developed. This could be further informed as part of the authorisation process and augmented via a publicly available register or database. Thirdly, further specialist research and development, allied with laboratory-based evidence, is required to evaluate the potential of using a weight of evidence approach for the design and development of detection methods for PBOs. This concept centres on using other indicators, aside from the single mutation of interest, to increase the likelihood of providing a unique signature or footprint. This includes consideration of the genetic background, flanking regions, off-target mutations, potential CRISPR/Cas activity, feasibility of heritable epigenetic and epitranscriptomic changes, as well as supplementary material from supplier, origin, pedigree and other documentation. Fourthly, additional work is recommended, evaluating the extent/type/nature of the genetic changes, and assessing the feasibility of applying threshold limits associated with these genetic changes to make any distinction on how they may have occurred. Such a probabilistic approach, supported with bioinformatics, to determine the likelihood of particular changes occurring through genome editing or traditional processes, could facilitate rapid classification and pragmatic labelling of products and organisms containing specific mutations more readily. Finally, several scientific publications on detection of genome edited products have been based on theoretical principles. It is recommended to further qualify these using evidenced based practical experimental work in the laboratory environment. Additional challenges and recommendations regarding the design, development and implementation of potential detection methods were also identified. Modern molecular biology-based techniques, inclusive of qPCR, dPCR, and NGS, in combination with appropriate bioinformatics pipelines, continue to offer the best analytical potential for developing methods for detecting PBOs. dPCR and NGS may offer the best technical potential, but qPCR remains the most practicable option as it is embedded in most analytical laboratories. Traditional screening approaches, similar to those for conventional transgenic GMOs, cannot easily be used for PBOs due to the deficit in common control elements incorporated into the host genome. However, some limited screening may be appropriate for PBOs as part of a triage system, should a priori information be known regarding the sequences of interest. The current deficit of suitable methods to detect and identify PBOs precludes accurate PBO quantification. Development of suitable reference materials to aid in the traceability of PBOs remains an issue, particularly for those PBOs which house on- and off-target mutations which can segregate. Off-target mutations may provide an additional tool to augment methods for detection, but unless these exhibit complete genetic linkage to the sequence of interest, these can also segregate out in resulting generations. Further research should be conducted regarding the likelihood of multiple mutations segregating out in a PBO, to help inform the development of appropriate PBO reference materials, as well as the potential of using off-target mutations as an additional tool for PBO traceability. Whilst recognising the technical challenges of developing and maintaining pan-genomic databases, this report recommends that the UK continues to consider development of such a resource, either as a UK centric version, or ideally through engagement in parallel EU and international activities to better achieve harmonisation and shared responsibilities. Such databases would be an invaluable resource in the design of reliable detection methods, as well as for confirming that a mutation is as a result of genome editing. PBOs and their products show great potential within the agri-food sector, necessitating a science-based analytical framework to support UK legislation, business and consumers. Differentiating between PBOs generated through genome editing compared to organisms which exhibit the same mutational change through traditional processes remains analytically challenging, but a broad set of diagnostic technologies (e.g., qPCR, NGS, dPCR) coupled with pan-genomic databases and bioinformatics approaches may help contribute to filling this analytical gap, and support the safety, transparency, proportionality, traceability and consumer confidence associated with the UK food chain.
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