Journal articles: 'CRISPR/Cas 9'

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 (May 31, 2020): 82–94. http://dx.doi.org/10.46501/ijmtst060515.

Full text

Abstract:

“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).

APA, Harvard, Vancouver, ISO, and other styles

2

Mali, Franc. "Is the Patent System the Way Forward with the CRISPR-Cas 9 Technology?" Science & Technology Studies 33, no. 4 (January 15, 2020): 2–23. http://dx.doi.org/10.23987/sts.70114.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

3

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 (March 1, 2017): G257—G265. http://dx.doi.org/10.1152/ajpgi.00410.2016.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

4

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.

Full text

APA, Harvard, Vancouver, ISO, and other styles

5

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

6

Hoffmann, Mareike D., Sabine Aschenbrenner, Stefanie Grosse, Kleopatra Rapti, Claire Domenger, Julia Fakhiri, Manuel Mastel, et al. "Cell-specific CRISPR–Cas9 activation by microRNA-dependent expression of anti-CRISPR proteins." Nucleic Acids Research 47, no. 13 (April 15, 2019): e75-e75. http://dx.doi.org/10.1093/nar/gkz271.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

7

Dayan, Fazli. "CRISPR Cas-9 genome editing and Islam: A religious perspective." Bangladesh Journal of Medical Science 18, no. 1 (December 30, 2018): 7–13. http://dx.doi.org/10.3329/bjms.v18i1.39540.

Full text

Abstract:

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

APA, Harvard, Vancouver, ISO, and other styles

8

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.

Full text

APA, Harvard, Vancouver, ISO, and other styles

9

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 (November 22, 2013): 994–1001. http://dx.doi.org/10.1128/aem.03015-13.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

10

Siew, Wei Sheng, Yin Quan Tang, Chee Kei Kong, Bey-Hing Goh, Serena Zacchigna, Kamal Dua, Dinesh Kumar Chellappan, et al. "Harnessing the Potential of CRISPR/Cas in Atherosclerosis: Disease Modeling and Therapeutic Applications." International Journal of Molecular Sciences 22, no. 16 (August 5, 2021): 8422. http://dx.doi.org/10.3390/ijms22168422.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

11

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 (September 27, 2018): e2009. http://dx.doi.org/10.1002/rmv.2009.

Full text

APA, Harvard, Vancouver, ISO, and other styles

12

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

13

Ratan, Zubair Ahmed, Young-Jin Son, Mohammad Faisal Haidere, Bhuiyan Mohammad Mahtab Uddin, Md Abdullah Yusuf, Sojib Bin Zaman, Jong-Hoon Kim, Laila Anjuman Banu, and Jae Youl Cho. "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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

14

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

15

Zhu, Haocheng, Chao Li, and Caixia Gao. "Applications of CRISPR–Cas in agriculture and plant biotechnology." Nature Reviews Molecular Cell Biology 21, no. 11 (September 24, 2020): 661–77. http://dx.doi.org/10.1038/s41580-020-00288-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

16

Değirmenci, Laura, Dietmar Geiger, Fábio Luiz Rogé Ferreira, Alexander Keller, Beate Krischke, Martin Beye, Ingolf Steffan-Dewenter, and Ricarda Scheiner. "CRISPR/Cas 9-Mediated Mutations as a New Tool for Studying Taste in Honeybees." Chemical Senses 45, no. 8 (September 24, 2020): 655–66. http://dx.doi.org/10.1093/chemse/bjaa063.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

17

Dayan, Fazli. "Ethico-legal aspects of CRISPR Cas-9 genome editing: A balanced approach." Bangladesh Journal of Medical Science 19, no. 1 (November 3, 2019): 11–16. http://dx.doi.org/10.3329/bjms.v19i1.43869.

Full text

Abstract:

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

APA, Harvard, Vancouver, ISO, and other styles

18

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.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

19

Singh, Desh D., Ravi Verma, Piyush Parimoo, Ashish Sahu, Vikram Kumar, Era Upadhyay, and Dharmendra K. Yadav. "Potential Therapeutic Relevance of CRISPR/Cas9 Guided Epigenetic Regulations for Neuropsychiatric Disorders." Current Topics in Medicinal Chemistry 21, no. 10 (June 17, 2021): 878–94. http://dx.doi.org/10.2174/1568026621666210317154502.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

20

Barakat, R. H., D. A. Habashy, A. Bahaa, and H. Adwan. "Impact of CCL4 knockout using CRISPR Cas-9 technology on colorectal tumour progression." Annals of Oncology 30 (October 2019): v242. http://dx.doi.org/10.1093/annonc/mdz246.120.

Full text

APA, Harvard, Vancouver, ISO, and other styles

21

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 (February 18, 2020): 467. http://dx.doi.org/10.3390/cells9020467.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

22

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 (October 22, 2018): i3—i9. http://dx.doi.org/10.1093/femsle/fny257s.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

23

Li, Rui, Xianyou Xia, Xing Wang, Xiaoyu Sun, Zhongye Dai, Dawei Huo, Huimin Zheng, Haiqing Xiong, Aibin He, and Xudong Wu. "Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model." PLOS Biology 18, no. 11 (November 30, 2020): e3000749. http://dx.doi.org/10.1371/journal.pbio.3000749.

Full text

Abstract:

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

APA, Harvard, Vancouver, ISO, and other styles

24

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 (June 8, 2018): 56. http://dx.doi.org/10.26694/jibi.v3i1.6258.

Full text

Abstract:

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.

APA, Harvard, Vancouver, ISO, and other styles

25

Behler, Juliane. "Kidnapping der wirtseigenen Nuklease RNase E durch ein CRISPR-Cas-System." BIOspektrum 25, no. 7 (November 2019): 790–91. http://dx.doi.org/10.1007/s12268-019-1310-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

26

Man, Dula, Brett Sansbury, Pawel Bialk, Kevin Bloh, E. Anders Kolb, and Eric Brian Kmiec. "Target Site Mutagenesis during Crispr/ Cas 9/Single-Stranded- Oligonucleotide Directed Gene Editing for Sickle Cell Anemia." Blood 128, no. 22 (December 2, 2016): 4706. http://dx.doi.org/10.1182/blood.v128.22.4706.4706.

Full text

Abstract:

Abstract Introduction Sickle Cell Disease (SCD) results from a simple substitution of valine for glutamic acid at codon 6 in the β globin gene, resulting in an AàT transition in the third position. The mutation results in the production of hemoglobin HbS which differs from the normal HbAin that it tends to polymerize into long strands that deform the erythrocyte. While a variety of traditional treatment regimens or reagents, such as hydroxyurea and chronic transfusions have been used widely, these therapies are wrought with short and long term side effects that limit efficacy. There is great interest in the hypothesis that the repair of a single nucleotide is facilitated by the combined action of CRISPR/Cas 9 and single-stranded oligonucleotides (ssODNs) could prove a significant therapeutic advance for sickle cell disease. CRISPR/Cas 9 induces a site-specific double-stranded break while the single-stranded oligonucleotide provides a DNA template to improve the rate of accurate genetic correction. There is a great effort to understand off-site mutagenesis caused by editing of DNA regions remote to the target sequence. It is critical that investigators understand the frequency and types of DNA alterations induced by the gene editing reaction and affecting the region surrounding the targeted nucleotide site (herein termed on-site mutagenesis). Methods We targeted beta globin genes with a CRISPR/Cas9 system designed to cleave 2 bases to the 5'side of the targeted (A) nucleotide (the PAM site is located 2 bases to the 3' side on the complimentary strand) coupled to the electroporation of a single-stranded oligonucleotide (72-mer) designed to convert the wild-type (A) to the mutant (T) nucleotide. To evaluate the rate of on-site mutagenesis among individual alleles, we clonally expanded populations of edited K562 cells. We hypothesize that the evaluation of individual clones will permit a clear identification of intended and un-intended on-site DNA alteration. Allelic heterogeneity is analyzed using Tracking of Indels by DEcompositon (TIDE) methodology combined with Sanger sequencing. Results We isolated 26 clonal lines for continued expansion after evidence of CRISPR/Cas 9 plasmid uptake and activity. Sanger sequencing with TIDE analysis revealed that the DNA sequence surrounding the targeted base is altered significantly as a result of the CRISPR/Cas9 gene editing process. Twenty three percent of the clones contain at least one corrected allele but one hundred percent of the clones exhibited mutagenicity of the DNA sequence surrounding the targeted base. All clones analyzed displayed varying degrees of sequence alteration (Figure 1). Interestingly, one clone contained a DNA insertion homologous to a region of the delta globin gene, suggesting that gene editing of the beta globin gene may have been repaired, in part, by delta globin DNA. The sequence of the delta globin locus was not changed. Conclusions Taken together, our data suggest that combinatorial approaches to beta globin gene editing using CRISPR/Cas 9 and single-stranded oligonucleotides induce significant onsite mutagenesis and potential genetic swapping between related members of the same gene family. These observations provide insight into the type of molecular activity that accompanies combinatorial gene editing, particularly surrounding the target site. This work is supported by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number P20GM109021 Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

27

Vaishnav, Radhika A. "Nanobyte." International Journal of Molecular and Immuno Oncology 3, no. 2 (July 25, 2018): 36. http://dx.doi.org/10.18203/issn.2456-3994.intjmolimmunooncol20183224.

Full text

Abstract:

An unintended effect of CRISPR-Cas-9 gene therapy may be the natural selection of p53-deficient cells. While this was unexpected, it highlights the need for continued basic research and carefully designed clinical trials to evaluate the mechanism and safety of this outcome.

APA, Harvard, Vancouver, ISO, and other styles

28

Anzalone, Andrew V., Luke W. Koblan, and David R. Liu. "Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors." Nature Biotechnology 38, no. 7 (June 22, 2020): 824–44. http://dx.doi.org/10.1038/s41587-020-0561-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

29

Stoll, Britta, Lisa-Katharina Maier, Sita J. Lange, Jutta Brendel, Susan Fischer, Rolf Backofen, and Anita Marchfelder. "Requirements for a successful defence reaction by the CRISPR–Cas subtype I-B system." Biochemical Society Transactions 41, no. 6 (November 20, 2013): 1444–48. http://dx.doi.org/10.1042/bst20130098.

Full text

Abstract:

Uptake of foreign mobile genetic elements is often detrimental and can result in cell death. For protection against invasion, prokaryotes have developed several defence mechanisms, which take effect at all stages of infection; an example is the recently discovered CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) immune system. This defence system directly degrades invading genetic material and is present in almost all archaea and many bacteria. Current data indicate a large variety of mechanistic molecular approaches. Although almost all archaea carry this defence weapon, only a few archaeal systems have been fully characterized. In the present paper, we summarize the prerequisites for the detection and degradation of invaders in the halophilic archaeon Haloferax volcanii. H. volcanii encodes a subtype I-B CRISPR–Cas system and the defence can be triggered by a plasmid-based invader. Six different target-interference motifs are recognized by the Haloferax defence and a 9-nt non-contiguous seed sequence is essential. The repeat sequence has the potential to fold into a minimal stem–loop structure, which is conserved in haloarchaea and might be recognized by the Cas6 endoribonuclease during the processing of CRISPR loci into mature crRNA (CRISPR RNA). Individual crRNA species were present in very different concentrations according to an RNA-Seq analysis and many were unable to trigger a successful defence reaction. Recognition of the plasmid invader does not depend on its copy number, but instead results indicate a dependency on the type of origin present on the plasmid.

APA, Harvard, Vancouver, ISO, and other styles

30

Bergel, Salvador Darío. "El impacto ético de las nuevas tecnologías de edición genética." Revista Bioética 25, no. 3 (December 2017): 454–61. http://dx.doi.org/10.1590/1983-80422017253202.

Full text

Abstract:

Resumen El descubrimiento de la técnica CRISPR/CAS 9 de edición genética abre importantes horizontes para la investigación científica. Los problemas éticos, jurídicos y sociales que pueden importar su aplicación a humanos son inmensos, lo que justifica un amplio debate social. El trabajo indaga sobre los temas más significativos que podría incluir tal debate.

APA, Harvard, Vancouver, ISO, and other styles

31

Littler, S., H. Whalley, S. Sousa, and S. Taylor. "CRISPR/Cas-9-mediated targeting of TP53 and MYC to investigate antimitotic mode of action." European Journal of Cancer 61 (July 2016): S24. http://dx.doi.org/10.1016/s0959-8049(16)61073-0.

Full text

APA, Harvard, Vancouver, ISO, and other styles

32

Maria Hupffer, Haide, and Juliane Altmann Berwig. "A tecnologia CRISPR-CAS 9: da sua compreensão aos desafios éticos, jurídicos e de governança." Revista Pensar 25, no. 3 (2020): 1–16. http://dx.doi.org/10.5020/2317-2150.2018.9722.

Full text

APA, Harvard, Vancouver, ISO, and other styles

33

Al-Sammarraie, Nadia, and Swapan K. Ray. "Applications of CRISPR-Cas9 Technology to Genome Editing in Glioblastoma Multiforme." Cells 10, no. 9 (September 7, 2021): 2342. http://dx.doi.org/10.3390/cells10092342.

Full text

Abstract:

Glioblastoma multiforme (GBM) is an aggressive malignancy of the brain and spinal cord with a poor life expectancy. The low survivability of GBM patients can be attributed, in part, to its heterogeneity and the presence of multiple genetic alterations causing rapid tumor growth and resistance to conventional therapy. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated (Cas) nuclease 9 (CRISPR-Cas9) system is a cost-effective and reliable gene editing technology, which is widely used in cancer research. It leads to novel discoveries of various oncogenes that regulate autophagy, angiogenesis, and invasion and play important role in pathogenesis of various malignancies, including GBM. In this review article, we first describe the principle and methods of delivery of CRISPR-Cas9 genome editing. Second, we summarize the current knowledge and major applications of CRISPR-Cas9 to identifying and modifying the genetic regulators of the hallmark of GBM. Lastly, we elucidate the major limitations of current CRISPR-Cas9 technology in the GBM field and the future perspectives. CRISPR-Cas9 genome editing aids in identifying novel coding and non-coding transcriptional regulators of the hallmarks of GBM particularly in vitro, while work using in vivo systems requires further investigation.

APA, Harvard, Vancouver, ISO, and other styles

34

Sampson, Timothy R., Sunil D. Saroj, Anna C. Llewellyn, Yih-Ling Tzeng, and David S. Weiss. "Author Correction: A CRISPR/Cas system mediates bacterial innate immune evasion and virulence." Nature 570, no. 7760 (May 24, 2019): E30—E31. http://dx.doi.org/10.1038/s41586-019-1253-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

35

Hahn, Florian, and Vladimir Nekrasov. "CRISPR/Cas precision: do we need to worry about off-targeting in plants?" Plant Cell Reports 38, no. 4 (November 13, 2018): 437–41. http://dx.doi.org/10.1007/s00299-018-2355-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

36

Capdeville, Niklas, Patrick Schindele, and Holger Puchta. "Application of CRISPR/Cas-mediated base editing for directed protein evolution in plants." Science China Life Sciences 63, no. 4 (March 5, 2020): 613–16. http://dx.doi.org/10.1007/s11427-020-1655-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

37

De, Aniket, and Dr Arup Ratan Biswas. "Elucidative PAM/Target Sequence for CRISPR/Cas- 9 Activity in Breast Cancer Using a Computational Approach." International Journal of Innovative Science and Research Technology 5, no. 7 (August 2, 2020): 872–76. http://dx.doi.org/10.38124/ijisrt20jul757.

Full text

Abstract:

Directed genomic area for release and modification are indicated by the nearness of nuclease explicit PAM sequence. By hindering the signs, Herceptin can slow or stop bosom malignant growth development. The outcomes show 4 top of the line direct RNAs for altering HER2 quality, the objective arrangements that are pertinent for cleavage by that gRNA, likewise 4. The central clarification for the terrible sign of cockeyed cleavage is accepted to be in the arranging of the single guide RNA for the Cas-9 protein. This is extremely basic for researchers to know while planning guide RNA. Albeit the two guys and females are inclined to bosom malignant growth, it is more probable for females to build up this sort of disease. Be that as it may, with such a large number of HER2 receptors, bosom malignant growth cells can assemble an excessive number of development signals. The most widely recognized qualities engaged with bosom malignancy incorporate HER2, BRCA1, and BRCA2. In this manner, we propose the genomic approach of controlling the HER2 quality guided by CRISPR/Cas-9 to guarantee lesser symptoms and increasingly powerful treatment.

APA, Harvard, Vancouver, ISO, and other styles

38

Meiliana, Anna, Nurrani Mustika Dewi, and Andi Wijaya. "Genome Editing with Crispr-Cas9 Systems: Basic Research and Clinical Applications." Indonesian Biomedical Journal 9, no. 1 (April 1, 2017): 1. http://dx.doi.org/10.18585/inabj.v9i1.272.

Full text

Abstract:

BACKGROUND: Recently established genome editing technologies will open new avenues for biological research and development. Human genome editing is a powerful tool which offers great scientific and therapeutic potential.CONTENT: Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPRassociated protein 9 (Cas9) technology is revolutionizing the gene function studies and possibly will give rise to an entirely new degree of therapeutics for a large range of diseases. Prompt advances in the CRISPR/Cas9 technology, as well as delivery modalities for gene therapy applications, are dismissing the barriers to the clinical translation of this technology. Many studies conducted showed promising results, but as current available technologies for evaluating off-target gene modification, several elements must be addressed to validate the safety of the CRISPR/Cas9 platform for clinical application, as the ethical implication as well.SUMMARY: The CRISPR/Cas9 system is a powerful genome editing technology with the potential to create a variety of novel therapeutics for a range of diseases, many of which are currently untreatable.KEYWORDS: genome editing, CRISPR-Cas, guideRNA, DSB, ZFNs, TALEN

APA, Harvard, Vancouver, ISO, and other styles

39

Vöneky, Silja. "International Standard Setting in Biomedicine – Foundations and New Challenges." Volume 61 · 2018 61, no. 1 (June 20, 2019): 131–51. http://dx.doi.org/10.3790/gyil.61.1.131.

Full text

Abstract:

This article examines current challenges for a normative framework regulating biomedicine, including those arising from the use of big data and machine learning tools, and from the use of the CRISPR/Cas-9 technology, as for instance gene drives. The article focusses on the question of legitimate standard setting and takes into account both “hard” and “soft” law as well as private rule making. This includes international treaties and declarations in the area of human rights law and environmental law, such as the International Covenant on Civil and Political Rights, the Cartagena Protocol on Biosafety to the Convention on Biological Diversity, the Rio Declaration on Environment and Development, and, more specifically, the UNESCO Declaration on Bioethics and Human Rights. The author argues that, as instruments of biotechnology and biomedicine merge, international environmental law has to be interpreted in the light of human rights law. In order to adapt to new challenges, the article calls for a humanisation of international environmental law and, because of the ongoing disruptive technological development, argues that further legitimate standard setting is required. Keywords: Biomedicine, Biotechnology, Gene Drives, Standard Setting, CRISPR/Cas-9, Artificial Intelligence

APA, Harvard, Vancouver, ISO, and other styles

40

Connahs, Heidi, Jelle van Creij, Sham Tlili, Tirtha Banerjee, Timothy Saunders, and Antonia Monteiro. "Targeting two different exons of Distal-less using CRISPR cas-9 produces butterflies with opposite phenotypes." Mechanisms of Development 145 (July 2017): S105—S106. http://dx.doi.org/10.1016/j.mod.2017.04.277.

Full text

APA, Harvard, Vancouver, ISO, and other styles

41

Wilding-Steele, Tom, Quentin Ramette, Paul Jacottin, and Philippe Soucaille. "Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum." International Journal of Molecular Sciences 22, no. 7 (April 2, 2021): 3704. http://dx.doi.org/10.3390/ijms22073704.

Full text

Abstract:

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology—several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.

APA, Harvard, Vancouver, ISO, and other styles

42

Meliawati, Meliawati, Christoph Schilling, and Jochen Schmid. "Recent advances of Cas12a applications in bacteria." Applied Microbiology and Biotechnology 105, no. 8 (March 23, 2021): 2981–90. http://dx.doi.org/10.1007/s00253-021-11243-9.

Full text

Abstract:

Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-mediated genome engineering and related technologies have revolutionized biotechnology over the last decade by enhancing the efficiency of sophisticated biological systems. Cas12a (Cpf1) is an RNA-guided endonuclease associated to the CRISPR adaptive immune system found in many prokaryotes. Contrary to its more prominent counterpart Cas9, Cas12a recognizes A/T rich DNA sequences and is able to process its corresponding guide RNA directly, rendering it a versatile tool for multiplex genome editing efforts and other applications in biotechnology. While Cas12a has been extensively used in eukaryotic cell systems, microbial applications are still limited. In this review, we highlight the mechanistic and functional differences between Cas12a and Cas9 and focus on recent advances of applications using Cas12a in bacterial hosts. Furthermore, we discuss advantages as well as current challenges and give a future outlook for this promising alternative CRISPR-Cas system for bacterial genome editing and beyond. Key points • Cas12a is a powerful tool for genome engineering and transcriptional perturbation • Cas12a causes less toxic side effects in bacteria than Cas9 • Self-processing of crRNA arrays facilitates multiplexing approaches

APA, Harvard, Vancouver, ISO, and other styles

43

Shirali, Akio, Wilson Minter Huijsmans, and Matteo Sottocornola. "Letter to the Editor: Genetic Editing of Secretory Pathway of Penicillium Chrysogenum After Observation of Increased Secretory Rates in an Increased Stress Environment (Microgravity), a Research Proposal by High School Students in Dubai." Fine Focus 4, no. 2 (December 21, 2018): 163–69. http://dx.doi.org/10.33043/ff.4.2.163-169.

Full text

Abstract:

In this study, we aim to amplify the secretory pathway of Penicillium Chrysogenum within the ISS or similar simulated microgravity using the miniPCR and/or RTQ-PCR and then optimizing Penicillium Chrysogenum function using CRISPR cas-9 (Clustered Regularly Interspaced Short Palindromic Repeats), a new technology in the genetics which can help in gene alteration for better drug production. The secretory pathway of Penicillium Chrysogenum is controlled by genes pcbAB , pcbC and penDE

APA, Harvard, Vancouver, ISO, and other styles

44

Liu, Qing, Chun Wang, Xiaozhen Jiao, Huawei Zhang, Lili Song, Yanxin Li, Caixia Gao, and Kejian Wang. "Hi-TOM: a platform for high-throughput tracking of mutations induced by CRISPR/Cas systems." Science China Life Sciences 62, no. 1 (November 13, 2018): 1–7. http://dx.doi.org/10.1007/s11427-018-9402-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

45

Elliott, Esther K., Larisa M. Haupt, and Lyn R. Griffiths. "Mini review: genome and transcriptome editing using CRISPR-cas systems for haematological malignancy gene therapy." Transgenic Research 30, no. 2 (February 20, 2021): 129–41. http://dx.doi.org/10.1007/s11248-020-00232-9.

Full text

APA, Harvard, Vancouver, ISO, and other styles

46

Stankovic, Tatjana, Nicholas Davies, Louise J. Tee, Andrew D. Beggs, and Malcolm Taylor. "Identification of Novel Therapeutic Targets in Atm-Deficient Lymphomas Using a Whole Genome CRISPR/CAS-9 Screen." Blood 134, Supplement_1 (November 13, 2019): 1504. http://dx.doi.org/10.1182/blood-2019-129974.

Full text

Abstract:

ATM is a principal DNA damage response protein that synchronises a complex network of cellular responses to double stranded DNA breaks. ATM gene is recurrently mutated in a wide range of lymphoid malignancies, including B-cell chronic lymphocytic leukemia (CLL), T-prolymphocytic leukaemia (T-PLL), mantle cell lymphoma (MCL) and diffuse B cell lymphoma (DLBCL). ATM pathway is utilized by many DNA damaging agents and consequently inactivation of this pathway can lead to chemoresistance. Furthermore, in the absence of ATM tumour cells exhibit genomic instability that can lead to clonal selection and evolution even under current targeted treatments. Consequently there is clear need to understand dependency pathways in ATM-deficient tumours and apply tailored targeted therapies that will specifically eliminate those tumour cells. We have previously presented a novel murine model of ATM-deficiency that spontaneously generate lymphoid tumours, mostly DLBCL. These tumours have been successfully propagated both in recipient mice and in vitro, where several cell lines have been generated. Genome editing methods, such as CRISPR/CAS-9, permit the targeted disruption of specific genes. Protocols for genome wide screens have been developed based on this technology which can be used to identify genes that are essential for cellular survival. As such, these screens can be used to identify dependency pathways for tumours with specific genetic lesions. Using lentiviral transduction we established two cell lines that stably expressed CAS-9. We then performed a genome wide CRISPR screen using the GeCKO library to identify novel therapeutic targets in these Atm-deficient tumours. This library consists of 130,209 unique single guide RNA (sgRNAs), targetting 20,611 genes including 1176 miRNAs. A comparative analysis was performed of sgRNA drop-out following 15 cellular doublings. This revealed a number of pathways including those already known to be synthetically lethal with ATM deficiency, such as ATR and PARP. Pathway analysis of the top genes from this drop-out analysis identified oxidative phosphorylation, the spliceosome, ribosome biogenesis, N-glycan biosynthesis, pyrimidine metabolism and purine metabolism as the most significantly affected pathways. Furthermore, the drop-out screen revealed a number of miRNAs, including MiR-3470a, Mir-3971, MiR-669f and MiR-719. These data provide a unique molecular assessment of the dependency of ATM-deficient lymphomas and provide a number of novel putative therapeutic targets for treating such tumours. Disclosures No relevant conflicts of interest to declare.

APA, Harvard, Vancouver, ISO, and other styles

47

Zhao, Zhe, Rui-an Zhang, Ge-yi Fu, Ran Zhang, Yan-fang Nie, Cong Sun, and Min Wu. "The Complete Genome of Emcibacter congregatus ZYLT, a Marine Bacterium Encoding a CRISPR-Cas 9 Immune System." Current Microbiology 77, no. 5 (January 9, 2020): 762–68. http://dx.doi.org/10.1007/s00284-019-01867-6.

Full text

APA, Harvard, Vancouver, ISO, and other styles

48

Zhou, Shiwei, Honghao Yu, Xiaoe Zhao, Bei Cai, Qiang Ding, Yu Huang, Yaxin Li, et al. "Generation of gene-edited sheep with a defined Booroola fecundity gene (FecBB) mutation in bone morphogenetic protein receptor type 1B (BMPR1B) via clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) 9." Reproduction, Fertility and Development 30, no. 12 (2018): 1616. http://dx.doi.org/10.1071/rd18086.

Full text

Abstract:

Since its emergence, the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) 9 system has been increasingly used to generate animals for economically important traits. However, most CRISPR/Cas9 applications have been focused on non-homologous end joining, which results in base deletions and insertions, leading to a functional knockout of the targeted gene. The Booroola fecundity gene (FecBB) mutation (p.Q249R) in bone morphogenetic protein receptor type 1B (BMPR1B) has been demonstrated to exert a profound effect on fecundity in many breeds of sheep. In the present study, we successfully obtained lambs with defined point mutations resulting in a p.249Q > R substitution through the coinjection of Cas9 mRNA, a single guide RNA and single-stranded DNA oligonucleotides into Tan sheep zygotes. In the newborn lambs, the observed efficiency of the single nucleotide exchange was as high as 23.8%. We believe that our findings will contribute to improved reproduction traits in sheep, as well as to the generation of defined point mutations in other large animals.

APA, Harvard, Vancouver, ISO, and other styles

49

Xu, Q., D. J. Milner, and M. B. Wheeler. "144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein." Reproduction, Fertility and Development 33, no. 2 (2021): 180. http://dx.doi.org/10.1071/rdv33n2ab144.

Full text

Abstract:

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

APA, Harvard, Vancouver, ISO, and other styles

50

Xu, Q., D. J. Milner, and M. B. Wheeler. "144 Use of the CRISPR/CAS 9 system to produce porcine adipose-derived stem cells expressing enhanced green fluorescent protein." Reproduction, Fertility and Development 33, no. 2 (2021): 180. http://dx.doi.org/10.1071/rdv33n2ab144.

Full text

Abstract:

The goal of our project is to produce porcine adipose-derived stem cells (ASCs) stably expressing enhanced green fluorescent protein (eGFP) by using the clustered regularly interspaced short palindromic repeats (CRIPSR) technique. Fluorescent stem cells can facilitate the tracing and visualisation of stem cell migration, fusion, and participation in tissue regeneration after stem cell injection therapy, and represent a useful tool for tissue engineering research. The production of stem cells containing eGFP from ASCs using the CRISPR gene editing technique is able to reduce the time and labour requirement necessary for harvesting fluorescent cells from transgenic pigs. To generate fluorescent, edited cells, we utilised the ROSA 26 locus of pigs for insertion of the eGFP gene by homology-directed repair of Cas9-cleaved DNA at the ROSA 26 locus. The critical steps of producing stem cells expressing eGFP are (1) cloning of guide oligos into a Cas9 cutting vector and producing a repair template vector to insert GFP; (2) transfecting porcine stem cells with CRISPR plasmids; (3) cell sorting with flow cytometry to isolate colonies expressing GFP. A Rosa 26 Cas9-gRNA cutting vector was produced by cloning a guide RNA sequence into the vector backbone of plasmid pX458-GFP, and the donor vector was produced by the combination of the eGFP gene flanked with ROSA 26 genomic DNA inserted into plasmid pUC57. To isolate cells edited to contain the eGFP gene inserted into the ROSA-26 locus, we transfected 250,000 cells with a 1:1 mass mixture of Cas9-gRNA and eGFP repair plasmid using Lipofectamine STEM reagent (Invitrogen) in three trials. GFP+ cells were isolated by fluorescence-activated cell sorting, plated in 96-well plates, and monitored for colony growth and GFP expression. These trials produced an average of ∼70 colonies from sorting, and ∼1% GFP+ colonies. As pX458 drives expression of GFP as a marker for transfection, we hypothesised that we would potentially isolate more GFP+ edited colonies if we utilised a Cas9-gRNA cutting vector expressing mCherry and sorted for cells expressing both mCherry and GFP. This would allow enrichment of edited cells expressing GFP early after transfection, without interference of cells expressing GFP from the Cas9-gRNA vector alone. Utilising this method, we again obtained an average of ∼70 colonies from sorting, and 3% GFP+ colonies. Results were subjected to Student’s t-test. The comparisons were colonies/cell sorted and GFP+ colonies/cell sorted. All data were expressed as quadratic means+mean SE. When we compared groups, no differences were found for colonies/cell sorted: P=0.53 (1.11 E-03±9.16E-04 and 5.39 E-04±3.77 E-04, respectively, for green-green or red-green) and for GFP+ colonies/cell sorted: P=0.44 (1.94 E-05±2.15E-05 and 4.59 E-05±2.46 E-05, respectively, for green-green or red-green). In conclusion, our attempts to isolate ASC edited to express GFP have been successful, and our initial results suggest that utilising a dual fluorescent label sorting strategy does not enhance the number of GFP+ ASC colonies isolated. Future studies will verify that our GFP+ ASC retain normal stem cell properties.

APA, Harvard, Vancouver, ISO, and other styles

Journal articles on the topic 'CRISPR/Cas 9'

Create a spot-on reference in APA, MLA, Chicago, Harvard, and other styles