Academic literature on the topic 'CRISPR/Cas9 transfection'
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Journal articles on the topic "CRISPR/Cas9 transfection"
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Zhang, Zhen, Lei Xiong, Chao Xie, Lingling Shen, Xuanhao Chen, Min Ye, Linyang Sun, et al. "Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth." International Journal of Molecular Sciences 23, no. 21 (October 27, 2022): 13042. http://dx.doi.org/10.3390/ijms232113042.
Full textAbstract:
The CRISPR/Cas9 system is an efficient tool for reverse genetics validation, and the application of this system in the cell lines provides a new perspective on target gene analysis for the development of biotechnology tools. However, in the cell lines of diamondback moth, Plutella xylostella, the integrity of the CRISPR/Cas9 system and the utilization of this cell lines still need to be improved to ensure the application of the system. Here, we stabilize the transfection efficiency of the P. xylostella cell lines at different passages at about 60% by trying different transfection reagents and adjusting the transfection method. For Cas9 expression in the CRIPSPR/Cas9 system, we identified a strong endogenous promoter: the 217-2 promoter. The dual-luciferase and EGFP reporter assay demonstrated that it has a driving efficiency close to that of the IE1 promoter. We constructed pB-Cas9-Neo plasmid and pU6-sgRNA plasmid for CRISPR/Cas9 system and subsequent cell screening. The feasibility of the CRISPR/Cas9 system in P. xylostella cell lines was verified by knocking out endogenous and exogenous genes. Finally, we generated a transgenic Cas9 cell line of P. xylostella that would benefit future exploitation, such as knock-in and multi-threaded editing. Our works provides the validity of the CRISPR/Cas9 system in the P. xylostella cell lines and lays the foundation for further genetic and molecular studies on insects, particularly favoring gene function analysis.
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Akbaba, Hasan, Gulsah Erel-Akbaba, and Serif Senturk. "Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing." Nanomedicine 16, no. 12 (May 2021): 963–78. http://dx.doi.org/10.2217/nnm-2020-0412.
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Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.
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Lanjewar, S. N., and K. R. Bondioli. "205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells." Reproduction, Fertility and Development 30, no. 1 (2018): 243. http://dx.doi.org/10.1071/rdv30n1ab205.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.
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Nasri, Masoud, Perihan Mir, Benjamin Dannenmann, Diana Amend, Yun Xu, Anna Solovyeva, Sylwia Stefanczyk, et al. "A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs." Blood 132, Supplement 1 (November 29, 2018): 1108. http://dx.doi.org/10.1182/blood-2018-99-114844.
Full textAbstract:
Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescent labeling of the Cas9/gRNA ribonucleoprotein complex (CRISPR/Cas9-gRNA RNP). We designed crRNA for exon 1 of GADD45b gene, annealed this crRNA with transactivating crRNA (tracrRNA) to form gRNA and covalently introduced one fluorchrome agent (CX-rhodamine or fluorescein) per approximately every 20 nucleotides. HEK293FT cells, Jurkat T-ALL cell line, bone marrow CD34+ HSPCs, and iPSCs were transfected with fluorescently-labeled GADD45b CRISPR/Cas9-gRNA RNP by means of cathionic polymer based transfection reagent for HEK293FT cells and Lonza 4D nucleofection for Jurkat T-ALL cell line, CD34+ HSPCs, and iPSCs. We detected CX-rhodamine- or fluorescein intracellular signals 12 hours after transfection that disappeared approximately 48 hours post transfection. Transfection efficiency varied between 40 % and 80 %, depending on the cell type. Labeling did not affect integrity of crRNA/tracRNA duplex formation, gene editing efficiency and off-target activities of CRISPR/Cas9-gRNA RNP, as assessed by Sanger sequencing and TIDE assay of transfected HEK293FT cells, Jurkat cells, CD34+ HSPCs and human iPSCs. Using fluorescein- or CX-rhodamine signal of labeled CRISPR/Cas9-gRNA RNP, we sorted and enriched gene-edited cells. Gene modification efficiency in sorted cells was between 40 and 70 %, based on the cell type. Of note, we detected much lower transfection and editing efficiency of the fused Cas9-EGFP protein assembled with GADD45b targeting gRNA, as compared to CRISPR/Cas9-gRNA RNP. Most probably, conjugation of EGFP tag is affecting functions of CRISPR/Cas9- gRNA RNP. GADD45b (Growth Arrest And DNA Damage Inducible Beta), also termed myeloid differentiation primary response 118 gene (MyD118), belongs to a family of evolutionarily conserved GADD45 proteins (GADD45a, GADD45b and GADD45g) that function as stress sensors regulating cell cycle, survival and apoptosis in response to stress stimulus as ultraviolet (UV)-induced DNA damage and genotoxic stress. We further performed functional studies of the effect of GADD45b knockout on cell growth and sensitivity to UV-induced DNA damage. Remarkably, we detected severe diminished viability of GADD45b-deficient HEK293FT, Jurkat cells, iPSCs and CD34+ HSPCs as compared to control transfected cells. We also found markedly elevated susceptibility of GADD45b-deficient Jurkat cells, CD34+ HSPCs and iPSCs to UV induced DNA damage, as documented by elevated levels of γH2AX (pSer139). Based on these observations, we conclude that GADD45b knockout using transfection of cells with labeled GADD45b-targeting CRISPR/Cas9-gRNA RNP led to increased susceptibility to DNA damage. Moreover, GADD45b deficient iPSCs retained pluripotency, but they failed to differentiate to mature neutrophils in embryoid body (EB)-based culture. Taken together, this is the first report describing transfection and sorting of primary hematopoietic cells and iPSCs using fluorescently-labeled CRISPR/Cas9-RNP, which is simple, safe and efficient method, and therefore may strongly expand the therapeutic avenues for gene-edited cells. Disclosures No relevant conflicts of interest to declare.
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Moradi, Pardis, Akbar Hasanzadeh, Fatemeh Radmanesh, Saideh Rajai Daryasarei, Elaheh Sadat Hosseini, Jafar Kiani, Ali Shahbazi, et al. "Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells." Nanotechnology 33, no. 7 (November 26, 2021): 075104. http://dx.doi.org/10.1088/1361-6528/ac357a.
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Abstract An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5–10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.
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Cheng, Hao, Feng Zhang, and Yang Ding. "CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications." Pharmaceutics 13, no. 10 (October 9, 2021): 1649. http://dx.doi.org/10.3390/pharmaceutics13101649.
Full textAbstract:
The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.
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McAndrews, Kathleen M., Fei Xiao, Antonios Chronopoulos, Valerie S. LeBleu, Fernanda G. Kugeratski, and Raghu Kalluri. "Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer." Life Science Alliance 4, no. 9 (July 19, 2021): e202000875. http://dx.doi.org/10.26508/lsa.202000875.
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CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant KrasG12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.
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Aschenbrenner, Sabine, Stefan M. Kallenberger, Mareike D. Hoffmann, Adrian Huck, Roland Eils, and Dominik Niopek. "Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity." Science Advances 6, no. 6 (February 2020): eaay0187. http://dx.doi.org/10.1126/sciadv.aay0187.
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The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.
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Aguilar, Rocio, Javier Fierro, Joshua Perez, and Huanyu Dou. "OMRT-12. Nanoparticle-based CRISPR-Cas9 delivery for anti-glioblastoma immunotherapy." Neuro-Oncology Advances 3, Supplement_2 (July 1, 2021): ii9. http://dx.doi.org/10.1093/noajnl/vdab070.036.
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Abstract Anti-glioblastoma GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) is an immune checkpoint that mediated the immune resistance. Inhibition of PD-L1 by antibodies was widely studied to treat many type of cancers. However, the inefficient therapeutic immune response became a significant barrier for treatment of GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. It is extremely difficulty to deliver CRISPR/Cas9 containing plasmid for translational and clinic applications. We have been developed a core-shell nanoparticle (NP) to carry CRISPR/Cas9 plasmid for PD-L1 knockout. The different NP formulations were made and optimized to deliver CRISPR/Cas9 plasmid. NPs were prepared by modifying the water temperature, sonication power and time and formulation time. The obtained NPs had a size of 115-160nm and a charge of 40-50mV. The size and charge were significantly altered after CRISPR/Cas9 plasmids were loaded into NPs (Cas9-NPs). Agarose gel electrophoresis showed that CRISPR/Cas9 plasmids were fully encapsulated by NPs with 1 and 2 ug. The positive DNA bands occurred with 4ng, indicating the overloaded CRISPR/Cas9 plasmid. Fluorescence microscopy determined Cas9-NPs uptake by U87 cells under a time-dependent manner. GFP tagged Cas9-NPs were treated to U87 cells for transfection evaluation. The obtained different NPs delivery of CRISPR/Cas9 exhibited various transfection efficiencies in U87 cells. Visualization of intracellular Cas9-NPs showed increases in uptake by U87 cells from 0.5, 1, 2, and 4 hours. The greater nuclear accumulation of Cas9-NPs was seen at 24 hours. A western blot assay determined the success of PD-L1 deletion by Cas9-NPs in human GBM U87 cells. NPs-based CRISPR/Cas9 gene-editing system has great potential as an immunotherapeutic platform to treat GBM.
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Atanes, Patricio, Inmaculada Ruz-Maldonado, Ross Hawkes, Bo Liu, Shanta J. Persaud, and Stefan Amisten. "Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing." Cellular Physiology and Biochemistry 45, no. 2 (2018): 656–66. http://dx.doi.org/10.1159/000487159.
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Background/Aims: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 β-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. Methods: The optimal transfection reagent for use in MIN6 β-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 β-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 β-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. Results: JetPRIME® was the best candidate for MIN6 β-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 β-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-β and IFNγ. Conclusion: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 β-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to β-cell proliferation and apoptosis.
Dissertations / Theses on the topic "CRISPR/Cas9 transfection"
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Phillips, Kelsey. "CRISPR-Cas9 Transfection Optimization and Use in a Forward Genetic Screen to Identify Telomere Length Maintenance Genes." BYU ScholarsArchive, 2018. https://scholarsarchive.byu.edu/etd/7357.
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Mutations in the telomere length maintenance pathway can lead to a spectrum of diseases called telomere syndromes, however, the pathway is not fully understood and there may still be unknown components. We designed a forward genetic screen to identify new genes involved in telomere length maintenance. Of the top ranked genes, ZNF827, a zinc finger protein, is the most promising candidate gene. The possible discovery of a new component involved in telomere length maintenance increases our understanding of the pathway and opens new avenues of research. Recent advances in molecular biology techniques, such as the use of RNA-guided nuclease CRISPR associated protein 9 (Cas9), have made screens like this possible. Cas9 is a nuclease that uses a guide RNA(gRNA) to direct its endonuclease activity. The use of Cas9 has revolutionized the field of genome engineering, providing scientists with more efficient methods to knockout and modify genomes. We sought to optimize CRISPR-Cas9 genome editing to make it as widely accessible as possible. We compared plasmid, ribonucleoprotein (RNP), and RNA only lipid-mediated transfection of CRISPR-Cas9 into cell lines using a novel reporter system to measure genome editing efficiency. All methods were successful to some extent, however, RNP lipofection was the most efficient and has many advantages over other methods. We also found that short homology arms of 30-35bp on donor templates was able to mediate site specific editing. These methods should broaden the accessibility of CRISPR-Cas9 genome editing.
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Santos, Rafael Miyashiro Nunes dos. "Substituição gênica ortotópica de porco para humano baseada em CRISPR/Cas9 e recombinases para xenotransplante." Universidade de São Paulo, 2017. http://www.teses.usp.br/teses/disponiveis/5/5168/tde-14112017-153947/.
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Modelos humanizados de porco são muito importantes para pesquisa biomédica e desenvolvimento de novas drogas e tratamentos. Além de ser um melhor modelo para doenças humanas do que animais de menor porte devido sua maior semelhança fisiológica, anatômica, de metabolismo e tempo de vida, o modelo suíno ainda permite suprimento ilimitado de órgãos para transplante. Apesar dessas vantagens, a expressão gênica inconsistente de animais transgênicos tornam a criação e avaliação desses animais muito dispendiosas, imprevisível e não permite a comparação de resultados de animais diferentes de maneira apropriada. Nesse estudo descrevemos uma nova técnica utilizando o promoter endógeno para a geração de um protocolo de substituição de genes com padrão clonal (transplante clonal de genes) sem clonagem de células, preservando a expressão genética e sua regulação intactas. Esse protocolo é reprodutível e pode ser aplicado para mais de um alvo genético, permitindo geração rápida de linhas transgênicas de animais (14-20 dias) com potencial de se tornar o novo padrão para geração de animais transgênicos de grande porte SuínosHumanized pig models are very important for biomedical research, and drugs and treatment development. Not only it is a better model for diseases than smaller animals because of its closer physiology, anatomy, metabolism and life span, it also may provide unlimited organs for transplantation. In spite of all this advantages, inconsistent gene expression in transgenic animals make its generation and evaluation expensive, unpredictable and do not allow proper outcome comparison between different animals. In this report we describe a reproducible technique utilizing the endogenous promoter for generation of a clonal pattern gene replacement protocol (clonal gene transplant) without cell cloning, maintaining the normal gene expression and its regulation. This protocol is reproducible and applicable to more than one gene target, allowing fast generation of transgenic animals cell lines (as low as 14-20 days) and could become the new standard for transgenic large animal generation
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Paladini, L. "BIOLOGICAL SIGNIFICANCE OF ALTERATIONS IN BRCA1 AND BRCA2 GENES AND RESPONSE TO DNA DAMAGE AGENTS IN HEREDITARY BREAST CANCER." Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/488444.
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Background: Although the large number of studies investigating BRCA mutations and their clinical role in different populations and ethnicities, there is a lack of a systematic analysis on these alterations in Italian cohorts, including the analysis of Variants of Unknown biological and clinical Significance (VUS). Moreover, correct management of breast cancer patients tested positive for alterations in BRCA1 or BRCA2 genes is still controversial. We aimed to assess the biological and clinical relevance of BRCA alterations in a consecutive cohort of hereditary breast cancer patients, with particular attention to VUS.
Methods: Genetic and clinical data from 366 patients with familial history of breast cancer were reviewed. The association between clinical-pathological, molecular data, and breast cancer patient subgroups was assessed. BRCA1/2 and γ-H2AX expression levels were assessed by qRT-PCR and IHC for all tumors. In silico protein prediction models were computed for VUS with potential clinical significance. Cell proliferation and apoptosis assays for CRISPR/Ca9s-generated mutant MDA-MB-231 cell line were performed to evaluate the sensitivity of specific VUS to DNA damage agents.
Results: Overall, 73 breast cancer patients (20%) tested positive for BRCA1/2 alterations. BRCA1 and BRCA2 mutations were reported in 34 (46.5%) and 15 (20.5%) patients, respectively. Two patients (3%) showed two concurrent mutations in both genes. Twenty-two patients (30%) tested positive for VUS. Breast cancer family history and early onset of disease were significantly associated with BRCA1 (p < 0.001) and BRCA2 (p = 0.045 and p = 0.005) mutations. Triple-negative histotype, grading 3, and high Ki-67 levels were significantly associated with BRCA1 mutations (p < 0.001). Molecular, in silico and in vitro experiments confirmed the deleterious effect of BRCA1 c.5509T>C VUS, which was associated with significant high levels of DNA damage and greater sensitivity to Olaparib compared to Cisplatin treatment.
Conclusions: Our study supports the deleterious effect of the BRCA1 c.5509T>C VUS in hereditary breast cancer patients, and suggests that breast cancer patient carriers of this variant could benefit from an intense surveillance and from a single agent treatment with Olaparib avoiding various side effects of chemotherapy treatment.
Book chapters on the topic "CRISPR/Cas9 transfection"
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López-Márquez, Arístides, Ainhoa Martínez-Pizarro, Belén Pérez, Eva Richard, and Lourdes R. Desviat. "Modeling Splicing Variants Amenable to Antisense Therapy by Use of CRISPR-Cas9-Based Gene Editing in HepG2 Cells." In Methods in Molecular Biology, 167–84. New York, NY: Springer US, 2022. http://dx.doi.org/10.1007/978-1-0716-2010-6_10.
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AbstractThe field of splice modulating RNA therapy has gained new momentum with FDA approved antisense-based drugs for several rare diseases. In vitro splicing assays with minigenes or patient-derived cells are commonly employed for initial preclinical testing of antisense oligonucleotides aiming to modulate splicing. However, minigenes do not include the full genomic context of the exons under study and patients’ samples are not always available, especially if the gene is expressed solely in certain tissues (e.g. liver or brain). This is the case for specific inherited metabolic diseases such as phenylketonuria (PKU) caused by mutations in the liver-expressed PAH gene.Herein we describe the generation of mutation-specific hepatic cellular models of PKU using CRISPR/Cas9 system, which is a versatile and easy-to-use gene editing tool. We describe in detail the selection of the appropriate cell line, guidelines for design of RNA guides and donor templates, transfection procedures and growth and selection of single-cell colonies with the desired variant, which should result in the accurate recapitulation of the splicing defect.