Journal articles on the topic 'Cas9 transfection'
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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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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.
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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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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.
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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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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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Sarkar, M. K., R. Uppala, S. Shao, M. Kahlenberg, and J. Gudjonsson. "386 Autocrine IFN-k restricts CRISPR-Cas9 keratinocyte transfection." Journal of Investigative Dermatology 139, no. 5 (May 2019): S67. http://dx.doi.org/10.1016/j.jid.2019.03.462.
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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.
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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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Đorđević, Marija, Verica Paunović, Maja Jovanović Tucović, Anja Tolić, Jovana Rajić, Svetlana Dinić, Aleksandra Uskoković, et al. "Nucleofection as an Efficient Method for Alpha TC1-6 Cell Line Transfection." Applied Sciences 12, no. 15 (August 8, 2022): 7938. http://dx.doi.org/10.3390/app12157938.
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An efficient transfection is a crucial step for the introduction of epigenetic modification in host cells, and there is a need for an optimized transfection process for individual model systems separately. Mouse pancreatic αTC1-6 cells, which act as an attractive model system for epigenetic cell reprogramming and diabetes treatment, were transiently transfected with two different transfection methods: the chemical method with polyethyleneimine (PEI) and nucleofection as a physical transfection method. Flow cytometry and fluorescent microscopy examination of GFP expression showed that transfection efficiency was affected by the size of plasmids using both transfection methods. Subsequently, the Cas9 mRNA expression confirmed successful transfection with EpiCRISPR plasmid, whereas the cell physiology remained unchanged. The adjusted nucleofection protocol for αTC1-6 cells transfected with an EpiCRISPR mix of plasmids reached 71.1% of GFP-positive transfected cells on the fifth post-transfection day and proved to be much more efficient than the 3.8% GFP-positive PEI transfected cells. Modifying the protocol, we finally specify CM-156 program and SF 4D-Nucleofector X Solutions for Amaxa™ nucleofection as a method of choice for alpha TC1-6 cell line transfection.
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Liang, Xiquan, Jason Potter, Shantanu Kumar, Yanfei Zou, Rene Quintanilla, Mahalakshmi Sridharan, Jason Carte, et al. "Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection." Journal of Biotechnology 208 (August 2015): 44–53. http://dx.doi.org/10.1016/j.jbiotec.2015.04.024.
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Langereis, Martijn A., Huib H. Rabouw, Melle Holwerda, Linda J. Visser, and Frank J. M. van Kuppeveld. "Knockout of cGAS and STING Rescues Virus Infection of Plasmid DNA-Transfected Cells." Journal of Virology 89, no. 21 (August 26, 2015): 11169–73. http://dx.doi.org/10.1128/jvi.01781-15.
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It is well known that plasmid DNA transfection, prior to virus infection, negatively affects infection efficiency. Here, we show that cytosolic plasmid DNA activates the cGAS/STING signaling pathway, which ultimately leads to the induction of an antiviral state of the cells. Using a transient one-plasmid clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, we generated cGAS/STING-knockout cells and show that these cells can be infected after plasmid DNA transfection as efficiently as nontransfected cells.
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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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Su, Kuan-Chung, Mary-Jane Tsang, Neil Emans, and Iain M. Cheeseman. "CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses." Molecular Biology of the Cell 29, no. 20 (October 2018): 2370–77. http://dx.doi.org/10.1091/mbc.e18-04-0214.
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A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
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Beneke, Tom, Ross Madden, Laura Makin, Jessica Valli, Jack Sunter, and Eva Gluenz. "A CRISPR Cas9 high-throughput genome editing toolkit for kinetoplastids." Royal Society Open Science 4, no. 5 (May 2017): 170095. http://dx.doi.org/10.1098/rsos.170095.
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Clustered regularly interspaced short palindromic repeats (CRISPR), CRISPR-associated gene 9 (Cas9) genome editing is set to revolutionize genetic manipulation of pathogens, including kinetoplastids. CRISPR technology provides the opportunity to develop scalable methods for high-throughput production of mutant phenotypes. Here, we report development of a CRISPR-Cas9 toolkit that allows rapid tagging and gene knockout in diverse kinetoplastid species without requiring the user to perform any DNA cloning. We developed a new protocol for single-guide RNA (sgRNA) delivery using PCR-generated DNA templates which are transcribed in vivo by T7 RNA polymerase and an online resource (LeishGEdit.net) for automated primer design. We produced a set of plasmids that allows easy and scalable generation of DNA constructs for transfections in just a few hours. We show how these tools allow knock-in of fluorescent protein tags, modified biotin ligase BirA*, luciferase, HaloTag and small epitope tags, which can be fused to proteins at the N- or C-terminus, for functional studies of proteins and localization screening. These tools enabled generation of null mutants in a single round of transfection in promastigote form Leishmania major , Leishmania mexicana and bloodstream form Trypanosoma brucei ; deleted genes were undetectable in non-clonal populations, enabling for the first time rapid and large-scale knockout screens.
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Foreman, Hui-Chen Chang, Varvara Kirillov, Gabrielle Paniccia, Demetra Catalano, Trevor Andrunik, Swati Gupta, Laurie T. Krug, and Yue Zhang. "RNA-guided gene editing of the murine gammaherpesvirus 68 genome reduces infectious virus production." PLOS ONE 16, no. 6 (June 4, 2021): e0252313. http://dx.doi.org/10.1371/journal.pone.0252313.
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Epstein-Barr virus (EBV) and Kaposi sarcoma herpesvirus (KSHV) are cancer-causing viruses that establish lifelong infections in humans. Gene editing using the Cas9-guideRNA (gRNA) CRISPR system has been applied to decrease the latent load of EBV in human Burkitt lymphoma cells. Validating the efficacy of Cas9-gRNA system in eradicating infection in vivo without off-target effects to the host genome will require animal model systems. To this end, we evaluated a series of gRNAs against individual genes and functional genomic elements of murine gammaherpesvirus 68 (MHV68) that are both conserved with KSHV and important for the establishment of latency or reactivation from latency in the host. gRNA sequences against ORF50, ORF72 and ORF73 led to insertion, deletion and substitution mutations in these target regions of the genome in cell culture. Murine NIH3T3 fibroblast cells that stably express Cas9 and gRNAs to ORF50 were most resistant to replication upon de novo infection. Latent murine A20 B cell lines that stably express Cas9 and gRNAs against MHV68 were reduced in their reactivation by approximately 50%, regardless of the viral gene target. Lastly, co-transfection of HEK293T cells with the vector expressing the Cas9-MHV68 gRNA components along with the viral genome provided a rapid read-out of gene editing and biological impact. Combinatorial, multiplex MHV68 gRNA transfections in HEK293T cells led to near complete ablation of infectious particle production. Our findings indicate that Cas9-gRNA editing of the murine gammaherpesvirus genome has a deleterious impact on productive replication in three independent infection systems.
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Colin, Béatrice, Benoit Deprez, and Cyril Couturier. "High-Throughput DNA Plasmid Transfection Using Acoustic Droplet Ejection Technology." SLAS DISCOVERY: Advancing the Science of Drug Discovery 24, no. 4 (October 5, 2018): 492–500. http://dx.doi.org/10.1177/2472555218803064.
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The Labcyte Echo acoustic liquid handler allows accurate droplet ejection at high speed from a source well plate to a destination plate. It has already been used in various miniaturized biological assays, such as quantitative PCR (q-PCR), quantitative real-time PCR (q-RT-PCR), protein crystallization, drug screening, cell dispensing, and siRNA transfection. However, no plasmid DNA transfection assay has been published so far using this dispensing technology. In this study, we evaluated the ability of the Echo 550 device to perform plasmid DNA transfection in 384-well plates. Due to the high throughput of this device, we simultaneously optimized the three main parameters of a transfection process: dilution of the transfection reagent, DNA amount, and starting DNA concentration. We defined a four-step protocol whose optimal settings allowed us to transfect HeLa cells with up to 90% efficiency and reach a co-expression of nearly 100% within transfected cells in co-transfection experiments. This fast, reliable, and automated protocol opens new ways to easily and rapidly identify optimal transfection settings for a given cell type. Furthermore, it permits easy software-based transfection control and multiplexing of plasmids distributed on wells of a source plate. This new development could lead to new array applications, such as human ORFeome protein expression or CRISPR-Cas9-based gene function validation in nonpooled screening strategies.
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Sazonova, Margarita A., Anastasia I. Ryzhkova, Vasily V. Sinyov, Marina D. Sazonova, Zukhra B. Khasanova, Nadezhda A. Nikitina, Vasily P. Karagodin, Alexander N. Orekhov, and Igor A. Sobenin. "Creation of Cultures Containing Mutations Linked with Cardiovascular Diseases using Transfection and Genome Editing." Current Pharmaceutical Design 25, no. 6 (June 12, 2019): 693–99. http://dx.doi.org/10.2174/1381612825666190329121532.
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Objective: In this review article, we analyzed the literature on the creation of cultures containing mutations associated with cardiovascular diseases (CVD) using transfection, transduction and editing of the human genome. Methods: We described different methods of transfection, transduction and editing of the human genome, used in the literature. Results: We reviewed the researches in which the creation of сell cultures containing mutations was described. According to the literature, system CRISPR/Cas9 proved to be the most preferred method for editing the genome. We found rather promising and interesting a practically undeveloped direction of mitochondria transfection using a gene gun. Such a gun can direct a genetically-engineered construct containing human DNA mutations to the mitochondria using heavy metal particles. However, in human molecular genetics, the transfection method using a gene gun is unfairly forgotten and is almost never used. : Ethical problems arising from editing the human genome were also discussed in our review. We came to a conclusion that it is impossible to stop scientific and technical progress. It is important that the editing of the genome takes place under the strict control of society and does not bear dangerous consequences for humanity. To achieve this, the constant interaction of science with society, culture and business is necessary. Conclusion: he most promising methods for the creation of cell cultures containing mutations linked with cardiovascular diseases, were system CRISPR/Cas9 and the gene gun.
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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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Raes, Laurens, Melissa Pille, Aranit Harizaj, Glenn Goetgeluk, Jelter Van Hoeck, Stephan Stremersch, Juan C. Fraire, et al. "Cas9 RNP transfection by vapor nanobubble photoporation for ex vivo cell engineering." Molecular Therapy - Nucleic Acids 25 (September 2021): 696–707. http://dx.doi.org/10.1016/j.omtn.2021.08.014.
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Oh, Soyoung A., Akiko Seki, and Sascha Rutz. "Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells." Current Protocols in Immunology 124, no. 1 (October 18, 2018): e69. http://dx.doi.org/10.1002/cpim.69.
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Lin, Michelle I., Elizabeth J. Paik, Bibhu P. Mishra, Song Chou, Ying Zhang, Kaleigh Tomkinson, Michael A. Pettiglio, et al. "Re-Creating Hereditary Persistence of Fetal Hemoglobin (HPFH) to Treat Sickle Cell Disease (SCD) and β-Thalassemia." Blood 128, no. 22 (December 2, 2016): 4708. http://dx.doi.org/10.1182/blood.v128.22.4708.4708.
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Abstract Hereditary persistence of fetal hemoglobin, a naturally occurring condition that substantially ameliorates disease in SCD and β-thalassemia, is associated with genetic variation at the β-globin locus. Our strategy is to use the CRISPR-Cas9 system to re-create specific genetic variants associated with HPFH in CD34+ hematopoietic stem and progenitor cells (HSPCs) and demonstrate their causal relationship to elevated fetal hemoglobin (HbF) levels as a potential therapeutic strategy to treat SCD and β-thalassemia. The CRISPR-Cas9 system has been adapted to achieve site-directed DNA cleavage by guide RNAs (gRNAs) and the Cas9 endonuclease. We have identified highly potent single gRNAs (sgRNAs) that can target regions within the β-globin locus associated with HPFH. The sgRNAs, when combined and delivered as dual sgRNAs, resulted in deletions that mimic naturally occurring deletions associated with HPFH. We have also optimized transfection dose and ratio of these sgRNAs with either Cas9 mRNA or Cas9 protein into primary human CD34+ HSPCs from mobilized peripheral blood of healthy donors and can achieve greater than 85% transfection rate with minimal cell loss (85-95% viability). We found that sgRNAs delivered with Cas9 protein resulted in better cell viability than when delivered with Cas9 mRNA especially at higher doses, while attaining the same rates of editing efficiency. Lastly, erythroid differentiation of CD34+ HSPCs demonstrated significant increase in γ-globin gene expression level by qRT-PCR as well as HbF protein levels, measured by FACS and LC/MS. In conclusion, we have optimized sgRNA and Cas9 transfection into primary human CD34+ HSPCs. We have identified sgRNAs that are highly effective in targeting the β-globin locus and employed forward genetics to re-create genetic variants associated with HPFH in HPSCs. We have demonstrated the causal relationship of different HPFH genetic variants to elevation of HbF, and obtained comparative data on upregulation of HbF in erythroid cells differentiated from edited CD34+ HSPCs. Our findings provide a viable therapeutic strategy using CRISPR-Cas9 for the treatment of β-hemoglobinopathies. Disclosures Lin: CRISPR Therapeutics: Employment. Paik:CRISPR Therapeutics: Employment. Mishra:CRISPR Therapeutics: Employment. Chou:CRISPR Therapeutics: Employment. Zhang:CRISPR Therapeutics: Employment, Equity Ownership. Tomkinson:CRISPR Therapeutics: Employment. Pettiglio:CRISPR Therapeutics: Employment. Sanginario:CRISPR Therapeutics: Employment. Woo:CRISPR Therapeutics: Employment. Lee:CRISPR Therapeutics: Employment. Allen:CRISPR Therapeutics: Employment. Cradick:CRISPR Therapeutics: Employment. Chakraborty:CRISPR Therapeutics: Employment. Porteus:CRISPR Therapeutics: Consultancy, Equity Ownership. Mavilio:Adverum Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Orchard Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy, Research Funding. Cowan:CRISPR Therapeutics: Employment, Equity Ownership. Novak:CRISPR Therapeutics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Lundberg:CRISPR Therapeutics: Employment, Equity Ownership.
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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.
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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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Seki, Akiko, and Sascha Rutz. "Optimized RNP transfection for highly efficient CRISPR/Cas9-mediated gene knockout in primary T cells." Journal of Experimental Medicine 215, no. 3 (February 7, 2018): 985–97. http://dx.doi.org/10.1084/jem.20171626.
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CRISPR (clustered, regularly interspaced, short palindromic repeats)/Cas9 (CRISPR-associated protein 9) has become the tool of choice for generating gene knockouts across a variety of species. The ability for efficient gene editing in primary T cells not only represents a valuable research tool to study gene function but also holds great promise for T cell–based immunotherapies, such as next-generation chimeric antigen receptor (CAR) T cells. Previous attempts to apply CRIPSR/Cas9 for gene editing in primary T cells have resulted in highly variable knockout efficiency and required T cell receptor (TCR) stimulation, thus largely precluding the study of genes involved in T cell activation or differentiation. Here, we describe an optimized approach for Cas9/RNP transfection of primary mouse and human T cells without TCR stimulation that results in near complete loss of target gene expression at the population level, mitigating the need for selection. We believe that this method will greatly extend the feasibly of target gene discovery and validation in primary T cells and simplify the gene editing process for next-generation immunotherapies.
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Sousa, Diana A., Ricardo Gaspar, Celso J. O. Ferreira, Fátima Baltazar, Ligia R. Rodrigues, and Bruno F. B. Silva. "In Vitro CRISPR/Cas9 Transfection and Gene-Editing Mediated by Multivalent Cationic Liposome–DNA Complexes." Pharmaceutics 14, no. 5 (May 19, 2022): 1087. http://dx.doi.org/10.3390/pharmaceutics14051087.
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Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) gene-editing offers exciting new therapeutic possibilities for disease treatment with a genetic etiology such as cancer, cardiovascular, neuronal, and immune disorders. However, its clinical translation is being hampered by the lack of safe, versatile, and effective nonviral delivery systems. Herein we report on the preparation and application of two cationic liposome–DNA systems (i.e., lipoplexes) for CRISPR/Cas9 gene delivery. For that purpose, two types of cationic lipids are used (DOTAP, monovalent, and MVL5, multivalent with +5e nominal charge), along with three types of helper lipids (DOPC, DOPE, and monoolein (GMO)). We demonstrated that plasmids encoding Cas9 and single-guide RNA (sgRNA), which are typically hard to transfect due to their large size (>9 kb), can be successfully transfected into HEK 293T cells via MVL5-based lipoplexes. In contrast, DOTAP-based lipoplexes resulted in very low transfection rates. MVL5-based lipoplexes presented the ability to escape from lysosomes, which may explain the superior transfection efficiency. Regarding gene editing, MVL5-based lipoplexes achieved promising GFP knockout levels, reaching rates of knockout superior to 35% for charge ratios (+/−) of 10. Despite the knockout efficiency being comparable to that of Lipofectamine 3000® commercial reagent, the non-specific gene knockout is more pronounced in MVL5-based formulations, probably resulting from the considerable cytotoxicity of these formulations. Altogether, these results show that multivalent lipid-based lipoplexes are promising CRISPR/Cas9 plasmid delivery vehicles, which by further optimization and functionalization may become suitable in vivo delivery systems.
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Tran, An, Javier Fierro, and Huanyu Dou. "CSIG-11. TARGETING PD-L1 IN GLIOBLASTOMA USING NANOPARTICLE-BASED GENE EDITING." Neuro-Oncology 22, Supplement_2 (November 2020): ii29—ii30. http://dx.doi.org/10.1093/neuonc/noaa215.123.
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Abstract Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor with very poor prognosis, usually with a less than one year survival rate. Immunotherapy has shown promising therapeutic potentials in research and clinical application, however, GBM associated immunosuppressive microenvironment creates a significant barrier for effective anti-GBM immune responses. The immune checkpoint PD-1/PD-L1 pathways play a critical role in tumor-induced immunosupression to evade immune surveillance. Gene-editing tools targeting PD-1/PD-L1 pathway have gained increased research attention, however the major challenge is how to effectively deliver gene-editing tools without causing adverse effects for clinical translation. We have developed a nanoparticle (NP) delivery system from a low molecular weight PEI lipid shell and a PLGA core that can package PD-L1 gRNA-CRISPR/Cas9 plasmid to transfect human U87 glioma cells overexpressing PD-L1. PD-L1 gRNA-CRISPR/Cas9 plasmid is constructed by inserting single guide targeting PD-L1 sequence into GFP CRISPR/Cas9 plasmid for visualizing transfection efficacy. NP is labelled with Rhodamine 6G to monitor cellular uptake and trafficking. Fluorescence microscopy shows human U87 glioma cells can quickly uptake PD-L1 GFP-CRISPR/Cas9 plasmid-NPs within 2 hours as indicated by the Rhodamine 6G. GFP expression was obtained after 48 hours of transfection and maintained up to 7 days without causing toxicity. Western blot analysis confirmed successful knockdown of PD-L1 in U87 cells. These findings reveal that NPs made by a cationic branched PEI lipid shell and a PLGA core are non-toxic and efficient in delivering CRISPR/Cas9 system to U87 cells. Editing of pathological gene in human glioma cells with PD-L1 GFP-CRISPR/Cas9 plasmid using NPs as a delivery system may provide a novel immunotherapy platform to treat GBM.
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Fajrial, Apresio K., Qing Qing He, Nurul I. Wirusanti, Jill E. Slansky, and Xiaoyun Ding. "A review of emerging physical transfection methods for CRISPR/Cas9-mediated gene editing." Theranostics 10, no. 12 (2020): 5532–49. http://dx.doi.org/10.7150/thno.43465.
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Sarkar, M., R. Uppala, A. Tsoi, S. Shao, A. C. Billi, B. E. Perez White, A. Kidder, X. Xing, J. Kahlenberg, and J. E. Gudjonsson. "276 Autocrine IFN-κ restricts CRISPR-Cas9 Keratinocyte transfection through STING-APOBEC3G activation." Journal of Investigative Dermatology 140, no. 7 (July 2020): S34. http://dx.doi.org/10.1016/j.jid.2020.03.282.
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Nii, Takenobu, Hiroshi Kohara, Tomotoshi Marumoto, Tetsushi Sakuma, Takashi Yamamoto, and Kenzaburo Tani. "Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency." Blood 126, no. 23 (December 3, 2015): 2037. http://dx.doi.org/10.1182/blood.v126.23.2037.2037.
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Abstract Human pluripotent stem cells (hPSCs), such as human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), have the potential to self-renew indefinitely and differentiate into various cell types. hPSCs can differentiate into various stem or progenitor cell populations used for regenerative medicine and drug development. Newly developed genome editing technology has advanced the use of hPSCs for such purposes. However, to fully utilize hPSCs to achieve this goal, more efficient gene transfer methods under defined conditions are required. Development of efficient genome editing methods, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9), for use in hPSCs holds great promise in the fields of basic and clinical research. Among these methods, TALENs are more efficient and safer for use in hPSCs to achieve specific gene editing, as ZFNs had a low gene editing efficiency and CRISPR/Cas9 was accompanied by more severe off-target effects than TALENs. Electroporation is a widely used transfection method for hPSC genome editing; however, this method results in reduced cell viability and gene editing efficiency. In the past decade, various methods were developed for gene transfer into hPSCs; however, hPSCs form tightly packed colonies, making gene transfer difficult. In this study, we established a culture method of hPSCs at a single-cell-state to reduce cell density, and investigated gene transfection efficiency followed by gene editing efficiency. hPSCs cultured in a single-cell-state were transfected using non-liposomal transfection reagents with plasmid DNA driven by the human elongation factor 1-alpha 1 (EF1α) promoter or mRNA encoding enhanced green fluorescent protein (eGFP). The proportion of eGFP+ cells considerably increased in single-cell-state cultures (DNA: 95.80 ± 2.51%, mRNA: 99.70 ± 0.10%). Moreover, most of the cells were viable (control: 93.10 ± 0.40%, DNA: 83.40 ± 2.03%, mRNA: 86.71 ± 0.19%). The mean fluorescence intensity (MFI) was approximately three-fold higher than that in cells transfected by electroporation (electroporation (EPN): 6631 ± 992; transfection (TFN): 17933 ± 1595). eGFP expression was detected by fluorescence microscopy until day seven post-transfection. Our results also demonstrate an inverse correlation between cell density and transfection efficiency. To test whether transfection using this method affected the "stemness" of hPSCs, we examined SSEA4 and NANOG expression in eGFP-transfected cells by flow cytometry analysis. The percentage of both SSEA4+ and NANOG+ cells was greater than 90%. Moreover, transplantation of eGFP-transfected cells into immunodeficient mice led to the formation of teratomas. These results strongly suggested that single-cell-state hPSC culture improved transfection efficiency without inducing differentiation or loss of pluripotency. Moreover, we used our efficient transfection method to edit the hPSC genome using TALENs. We constructed a Platinum TALEN driven by the EF1α promoter targeting the adenomatous polyposis coli (APC) gene and analyzed the efficiency of gene editing using the Cel-1 assay. Our efficient transfection method induced mutations more efficiently than electroporation (Transfection: 11.1 ± 1.38%, Electroporation: 3.2 ± 0.89). These results showed that TALENs increased gene editing efficiency in single-cell-state hPSC cultures. Overall, our efficient hPSC transfection method using single-cell-state culture provides an excellent experimental system to investigate the full potential of hPSCs. We expect that this method may contribute to the fields of hPSC-based regenerative medicine and drug discovery. Disclosures No relevant conflicts of interest to declare.
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Pavese, Vera, Andrea Moglia, Silvia Abbà, Anna Maria Milani, Daniela Torello Marinoni, Elena Corredoira, Maria Teresa Martínez, and Roberto Botta. "First Report on Genome Editing via Ribonucleoprotein (RNP) in Castanea sativa Mill." International Journal of Molecular Sciences 23, no. 10 (May 20, 2022): 5762. http://dx.doi.org/10.3390/ijms23105762.
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Castanea sativa is an important tree nut species worldwide, highly appreciated for its multifunctional role, in particular for timber and nut production. Nowadays, new strategies are needed to achieve plant resilience to diseases, climate change, higher yields, and nutritional quality. Among the new plant breeding techniques (NPBTs), the CRISPR/Cas9 system represents a powerful tool to improve plant breeding in a short time and inexpensive way. In addition, the CRISPR/Cas9 construct can be delivered into the cells in the form of ribonucleoproteins (RNPs), avoiding the integration of exogenous DNA (GMO-free) through protoplast technology that represents an interesting material for gene editing thanks to the highly permeable membrane to DNA. In the present study, we developed the first protoplast isolation protocol starting from European chestnut somatic embryos. The enzyme solution optimized for cell wall digestion contained 1% cellulase Onozuka R-10 and 0.5% macerozyme R-10. After incubation for 4 h at 25 °C in dark conditions, a yield of 4,500,000 protoplasts/mL was obtained (91% viable). The transfection capacity was evaluated using the GFP marker gene, and the percentage of transfected protoplasts was 51%, 72 h after the transfection event. The direct delivery of the purified RNP was then performed targeting the phytoene desaturase gene. Results revealed the expected target modification by the CRISPR/Cas9 RNP and the efficient protoplast editing.
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Pei, Guangying. "Research status of CRISPR/Cas9 delivery system." Highlights in Science, Engineering and Technology 21 (December 4, 2022): 96–100. http://dx.doi.org/10.54097/hset.v21i.3143.
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Deriving from the adaptive immune system of prokaryotes, the emerging gene-editing technology CRISPR/Cas9, has become the most popular and powerful tool for the manipulation of genes at the molecular level. It has the advantages of easy construction, high efficiency along with good specificity. CRISPR/Cas9 system has been extensively applied to model constructions, clinical treatment and mechanisms of tumorigenesis. As a number of strategies for CRISPR/Cas9 system have been developed, this review systematically summarizes the commonly applied methods along with discussions on their advantages and limitations and different scenarios for specific applications. The basic delivery forms include plasmid DNA with sgRNA, mRNA with sgRNA and a complex of Cas9 protein and sgRNA, ribonucleoprotein (RNP). For the methods of delivery into cells, there are viral vectors and non-viral vectors, which further consist of physical and chemical methods. While the efficiency and rate of successful transfection vary in different strategies, they commonly suffer from off-target effects, mutagenesis, cytotoxicity and immunogenicity to different extents.
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Lee, Han Ki, Heui Min Lim, See-Hyoung Park, and Myeong Jin Nam. "Knockout of Hepatocyte Growth Factor by CRISPR/Cas9 System Induces Apoptosis in Hepatocellular Carcinoma Cells." Journal of Personalized Medicine 11, no. 10 (September 29, 2021): 983. http://dx.doi.org/10.3390/jpm11100983.
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Background: CRISPR/Cas9 system is a prokaryotic adaptive immune response system that uses noncoding RNAs to guide the Cas9 nuclease to induce site-specific DNA cleavage. Hepatocyte growth factor (HGF) is a well-known growth factor that plays a crucial role in cell growth and organ development. According to recent studies, it has been reported that HGF promoted growth of hepatocellular carcinoma (HCC) cells. Here, we investigated the apoptotic effects in HCC cells. Methods: Crispr-HGF plasmid was constructed using GeneArt CRISPR Nuclease Vector. pMex-HGF plasmid that targets HGF overexpressing gene were designed with pMex-neo plasmid. We performed real time-polymerase chain reaction to measure the expression of HGF mRNA. We performed cell counting assay and colony formation assay to evaluate cell proliferation. We also carried out migration assay and invasion assay to reveal the inhibitory effects of Crispr-HGF in HCC cells. Furthermore, we performed cell cycle analysis to detect transfection of Crispr-HGF induced cell cycle arrest. Collectively, we performed annexin V/PI staining assay and Western blot assay. Results: In Crispr-HGF-transfected group, the mRNA expression levels of HGF were markedly downregulated compared to pMex-HGF-transfected group. Moreover, Crispr-HGF inhibited cell viability in HCC cells. We detected that wound area and invaded cells were suppressed in Crispr-HGF-transfected cells. The results showed that transfection of Crispr-HGF induced cell cycle arrest and apoptosis in HCC cells. Expression of the phosphorylation of mitogen activated protein kinases and c-Met protein was regulated in Crispr-HGF-transfected group. Interestingly, we found that the expression of HGF protein in conditioned media significantly decreased in Crispr-HGF-transfected group. Conclusions: Taken together, we found that inhibition of HGF through transfection of Crispr-HGF suppressed cell proliferation and induced apoptotic effects in HCC Huh7 and Hep3B cells.
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Reymond, Jean-Louis. "Peptide Dendrimers: From Enzyme Models to Antimicrobials and Transfection Reagents." CHIMIA International Journal for Chemistry 75, no. 6 (June 30, 2021): 535–38. http://dx.doi.org/10.2533/chimia.2021.535.
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Aiming at studying cooperativity effects between amino acids in easily accessible protein models, we have explored the chemistry of peptide dendrimers, which we obtain as pure products by solid-phase peptide synthesis using a branching diamino acid such as lysine at every second or third position in a peptide sequence, followed by reverse-phase HPLC purification. This article reviews discoveries driven by combinatorial library synthesis and screening, including enantioselective esterase and aldolase enzyme models, cobalamin binding and peroxidase dendrimers, glycopeptide dendrimer biofilm inhibitors and their X-ray crystal structures as complexes with lectins, antimicrobial peptide dendrimers active against multidrug resistant Gram-negative bacteria, and transfection reagents for siRNA and CRISPR-Cas9 plasmid DNA. Latest developments include cheminformatics and artificial intelligence for exploring the peptide chemical space, and the principle of stereorandomization to understand the role of peptide chirality in activity.
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Bierle, Craig J., Kaitlyn M. Anderholm, Jian Ben Wang, Michael A. McVoy, and Mark R. Schleiss. "Targeted Mutagenesis of Guinea Pig Cytomegalovirus Using CRISPR/Cas9-Mediated Gene Editing." Journal of Virology 90, no. 15 (May 25, 2016): 6989–98. http://dx.doi.org/10.1128/jvi.00139-16.
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ABSTRACTThe cytomegaloviruses (CMVs) are among the most genetically complex mammalian viruses, with viral genomes that often exceed 230 kbp. Manipulation of cytomegalovirus genomes is largely performed using infectious bacterial artificial chromosomes (BACs), which necessitates the maintenance of the viral genome inEscherichia coliand successful reconstitution of virus from permissive cells after transfection of the BAC. Here we describe an alternative strategy for the mutagenesis of guinea pig cytomegalovirus that utilizes clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9)-mediated genome editing to introduce targeted mutations to the viral genome. Transient transfection and drug selection were used to restrict lytic replication of guinea pig cytomegalovirus to cells that express Cas9 and virus-specific guide RNA. The result was highly efficient editing of the viral genome that introduced targeted insertion or deletion mutations to nonessential viral genes. Cotransfection of multiple virus-specific guide RNAs or a homology repair template was used for targeted, markerless deletions of viral sequence or to introduce exogenous sequence by homology-driven repair. As CRISPR/Cas9 mutagenesis occurs directly in infected cells, this methodology avoids selective pressures that may occur during propagation of the viral genome in bacteria and may facilitate genetic manipulation of low-passage or clinical CMV isolates.IMPORTANCEThe cytomegalovirus genome is complex, and viral adaptations to cell culture have complicated the study of infectionin vivo. Recombineering of viral bacterial artificial chromosomes enabled the study of recombinant cytomegaloviruses. Here we report the development of an alternative approach using CRISPR/Cas9-based mutagenesis in guinea pig cytomegalovirus, a small-animal model of congenital cytomegalovirus disease. CRISPR/Cas9 mutagenesis can introduce the same types of mutations to the viral genome as bacterial artificial chromosome recombineering but does so directly in virus-infected cells. CRISPR/Cas9 mutagenesis is not dependent on a bacterial intermediate, and defined viral mutants can be recovered after a limited number of viral genome replications, minimizing the risk of spontaneous mutation.
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Lee, Myoung Hui, Jiyoung Lee, Seung A. Choi, Ye-Sol Kim, Okjae Koo, Seung Hee Choi, Woo Seok Ahn, Eun Yee Jie, and Suk Weon Kim. "Efficient genome editing using CRISPR–Cas9 RNP delivery into cabbage protoplasts via electro-transfection." Plant Biotechnology Reports 14, no. 6 (November 12, 2020): 695–702. http://dx.doi.org/10.1007/s11816-020-00645-2.
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Nugrahaningsih, Dwi Aris Agung, Eko Purnomo, Widya Wasityastuti, Ronny Martien, Nur Arfian, and Tety Hartatik. "BMPR2 Editing in Fibroblast NIH3T3 using CRISPR/Cas9 Affecting BMPR2 mRNA Expression and Proliferation." Indonesian Biomedical Journal 14, no. 1 (March 17, 2022): 45–51. http://dx.doi.org/10.18585/inabj.v14i1.1724.
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BACKGROUND: Bone Morphogenetic Protein Receptor II (BMPR2) deficiency is associated with the pathologic development of pulmonary vascular changes in Pulmonary Arterial Hypertension (PAH). Fibroblast is the most abundant cell in vascular. However, there is only a little information regarding the effect of BMPR2 deficiency in fibroblast. This study aims to understand the effect of BMPR2 deficiency in fibroblasts.METHODS: This study applied the CRISPR/Cas9 technique to edit BMPPR2 in NIH-3T3 cells. The transfection of CRISPR/Cas9 for BMPR2 editing into NIH-3T3 cells was done by using chitosan nanoparticles. The evaluation of BMPR2 and Transforming Growth Factor (TGF)-β mRNA expression was done using Quantitative real-time polymerase chain reaction. The assessment of edited NIH-3T3 cells proliferation was done using a scratch test assay.RESULTS: The BMPR2 mRNA expression of CRISPR/Cas9-edited group was lower than the untreated group. The proliferation of the CRISPR/Cas9-edited group was higher than the untreated group. The TGF-β mRNA expression of CRISPR/Cas9-edited and untreated groups was similar.CONCLUSION: BMPR2 deficiency in fibroblast increase the fibroblast ability to proliferate.KEYWORDS: BMPR2, PAH, fibroblast NIH-3T3, CRISPR/Cas9, proliferation
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Vans Landschoot, G., R. J. Bevacqua, R. Fernandez y Martin, F. A. Pereyra-Bonnet, and D. F. Salamone. "201 Testing of single guide RNAs, optimization of transfection, and selection systems for the generation of SRY knockout foetal fibroblast cells." Reproduction, Fertility and Development 31, no. 1 (2019): 225. http://dx.doi.org/10.1071/rdv31n1ab201.
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Obtaining male and female cells from a male could have important implications for endangered mammalian species and domestic animal production. Achieving this could constitute a proof of concept of the use of assisted reproduction technologies for the conservation of endangered species. In particular, SRY is the principal male sex determinant gene and is found on the short arm of the Y chromosome. With the introduction of nuclease-mediated genome editing technologies, such as the CRISPR/Cas9 system, it is possible to envision precise DNA targeting in this gene as strategy to manipulate the sex of cell lines. Based on the above, we tested the CRISPR/Cas9 system to later create knockout (KO) cell lines of the SRY gene in bovine. The aim of this work was (1) to test the efficiency of single guide (sg)RNAs designed to target the bovine SRY gene in HEK293T cell line, and (2) to optimize blasticidin concentrations and electroporation conditions in bovine fetal fibroblast (BFF). To test sgRNA efficiency, we used 3 sgRNA designed and tested over the sequence of bovine SRY gene (690bp). The efficiency of each sgRNA was evaluated in a heterologous way by using a modification of pCAG-EGxxFP (plasmid #50716, Addgene, Cambridge, MA, USA) in which we inserted the bovine SRY sequence (NCBI Reference Sequence: NC_016145.1), and that only results in green fluorescent protein (GFP) expression upon cutting by the CRISPR/Cas9 system. We transfected the HEK293T cell line with the following groups to assess the efficiency of 3 sgRNA: (1) btSRY1+Cas9+pCAG-EGSRYFP; (2) btSRY2+Cas9+pCAG-EGSRYFP; (3) btSRY3+Cas9+pCAG-EGSRYFP; (4) PU6-empty+Cas9+pCAG- EGSRYFP; (5) GFP-only control. In addition, 6 blasticidin concentrations (0, 1, 2.5, 5, 10, and 20µg mL−1) and 3 electroporation conditions (Tfx1, Tfx2, and Tfx3) were tested, both in BFF. Electroporation conditions were as follows: Tfx1=1.25 kV cm−1; number of pulses=3; electrode gap=4 mm; interval=100 ms; Tfx2=1.2 kV/cm; number of pulses=4; electrode gap=4 mm; interval=100 ms; and Tfx3=voltage: 350V, LV mode; pulse length=100 µs; number of pulses=4; electrode gap=4 mm; interval=100ms. Statistical analyses were performed using 2-tailed Mann-Whitney tests. Results for sgRNA efficiency, based on GFP expression by counting GFP+ cells under fluorescent microscopy showed that btSRY1 (17%) and btSRY2 (13%) worked significantly better than btSRY3 (1%). The blasticidin selection assay showed that 5, 10, and 20µg mL−1were significantly more lethal than 0, 1 and 2.5µg mL−1, by counting living cells in Neubauer chamber. We chose 5µg mL−1 as the concentration for future experiments. Last, the Tfx2 electroporation protocol (11.34%) was more efficient than the other 2 protocols tested (Tfx1 and Tfx3: 3.48 and 0.86%, respectively), based on the counting of GFP+ cells under fluorescent microscopy. Electroporation of BFF with btSRY1, btSRY2, or both and Cas9 using Tfx2 protocol and molecular characterisation of colonies are currently in progress with the ultimate objective of producing SRY knockout bovine embryos by somatic cell nuclear transfer.
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Tavares, K. C. S., C. R. Lazzarotto, S. G. Neto, L. T. Martins, L. H. Aguiar, C. E. M. Calderón, L. P. R. Teixeira, et al. "241 CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 ASSOCIATED WITH TRANSIENT DEPLETION OF NON-HOMOLOGOUS END-JOINING PATHWAY INCREASED GENE-TARGETING EFFICIENCY IN GOAT FIBROBLASTS." Reproduction, Fertility and Development 28, no. 2 (2016): 252. http://dx.doi.org/10.1071/rdv28n2ab241.
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The transgenic animal platform for the expression of recombinant proteins in the milk offers particularly attractive possibilities. The recent application of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 to promote precise genome modifications and DNA editing also allows the targeting of specific DNA sequences in embryos or cells in culture. In addition, the transient knockdown of the NHEJ pathway by RNAi has been shown to increase gene‐targeting (GT) rate in cultured cells (Bertolini et al. 2009 Mol. Biotechnol. 41, 106–114). The CRISPR/Cas9 system was used to target a 15-kb transgene construct into the ROSA26 locus in goat fetal fibroblast cells subjected to a transient RNAi‐induced depletion of the NHEJ Ku70 protein. A polycistronic expression vector was constructed by ligating the coding sequences of 3 antigenic proteins against Brucella abortus linked by self-processing 2A peptides under the regulation of the bovine α-lactalbumin promoter. The final vector also contained the neomycin resistance gene and left and right 2-kb arms homologous to the goat ROSA26 locus. A total of 2 × 105 fibroblast cells at passage 3 from a 50-day fetus were transfected using the Neon Transfection System (Invitrogen/ThermoFisher Scientific, Waltham, MA, USA), according to the following groups: mock control (M); vector-only (V); vector + RNAi against Ku70 (VR); vector + ROSA26‐CRISPR/Cas9 (VC); and vector + RNAi against Ku70 + ROSA26-CRISPR/Cas9 (VCR). After antibiotic selection, colonies were characterised for zygosity, transgene copy number, and off-targets. Mortality rates following cell transfection were 68, 78, 75, 83, and 90%, and the number of colonies after selection was 0, 13, 22, 5, and 8 for the M, V, VR, VC, and VCR groups, respectively. Gene targeting was detected only when the ROSA26‐CRISPR/Cas9 was combined to the vector (VC group, 1 in 22 colonies) or to the vector and RNAi against Ku70 (VCR group, 1 in 8 colonies), with a 2.8-fold increase in GT rate when associating the 3 components (VCR group). No CRISPR/Cas9 off-targets were detected in 7 different sequenced hot spots. One colony from the VC group, harboring a biallelic transgene knock-in, was chosen for use in goat cloning by SCNT following our established procedures (Martins et al. 2015 Reprod. Fertil. Dev. 27, 111). Four viable pregnancies (33.3%) were established, based on the ultrasonographic visualisation of the embryo and heartbeat on Day 26, after the transfer of 144 embryos to 12 female recipient does, demonstrating the developmental potential of the transgenic knock-in donor cells. However, pregnancies were lost up to Day 55 of pregnancy. Our preliminary data suggest that the combined cell transfection of gene target-specific CRISPR/Cas9 and RNAi to knockdown the NHEJ pathway is a viable and efficient approach to produce precise genetically modified goat donor cells carrying mono- and biallelic knock-ins of large size transgene constructs for use in cloning by SCNT. Cloning procedures are underway using biallelic knock-in somatic cells to obtain live offspring, which will be the first step to produce and test a recombinant subunit vaccine against B. abortus.
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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.
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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.
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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 textAbstract:
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.
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Meng, Ruirui, Chenchen Wang, Lihua Wang, Yanlong Liu, Qiuwen Zhan, Jiacheng Zheng, and Jieqin Li. "An efficient sorghum protoplast assay for transient gene expression and gene editing by CRISPR/Cas9." PeerJ 8 (October 13, 2020): e10077. http://dx.doi.org/10.7717/peerj.10077.
Full textAbstract:
Protoplasts are commonly used in genetic and breeding research. In this study, the isolation of sorghum protoplasts was optimized and applied to transient gene expression and editing by CRISPR/Cas9. The protoplast was most viable in 0.5 M mannitol, which was the highest of three concentrations after 48- and 72-hours treatments. Using this method we can derive an average of 1.6×106 cells which vary from 5 to 22 nm in size. The average transfection of the protoplasts was 68.5% using the PEG-mediated method. The subcellular assays located Sobic.002G279100-GFP and GFP proteins in the cell compartments as predicted bioinformatically. Two CRISPR/Cas9 plasmids were transfected into sorghum protoplasts to screen for an appropriate sgRNA for gene editing. One plasmid can correctly edit the target region using a single protoplast cell as template DNA. Our results indicated that the protoplast assays as optimized are suitable for transient gene expression and sgRNA screening in CRISPR/Cas9 gene editing procedures.
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Martens, Marie Christine, Janin Edelkamp, Christina Seebode, Mirijam Schäfer, Susanne Stählke, Saskia Krohn, Ole Jung, Hugo Murua Escobar, Steffen Emmert, and Lars Boeckmann. "Generation and Characterization of a CRISPR/Cas9-Mediated SNAP29 Knockout in Human Fibroblasts." International Journal of Molecular Sciences 22, no. 10 (May 18, 2021): 5293. http://dx.doi.org/10.3390/ijms22105293.
Full textAbstract:
Loss-of-function mutations in the synaptosomal-associated protein 29 (SNAP29) lead to the rare autosomal recessive neurocutaneous cerebral dysgenesis, neuropathy, ichthyosis, and keratoderma (CEDNIK) syndrome. SNAP29 is a soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein. So far, it has been shown to be involved in membrane fusion, epidermal differentiation, formation of primary cilia, and autophagy. Recently, we reported the successful generation of two mouse models for the human CEDNIK syndrome. The aim of this investigation was the generation of a CRISPR/Cas9-mediated SNAP29 knockout (KO) in an immortalized human cell line to further investigate the role of SNAP29 in cellular homeostasis and signaling in humans independently of animal models. Comparison of different methods of delivery for CRISPR/Cas9 plasmids into the cell revealed that lentiviral transduction is more efficient than transfection methods. Here, we reported to the best of our knowledge the first successful generation of a CRISPR/Cas9-mediated SNAP29 KO in immortalized human MRC5Vi fibroblasts (c.169_196delinsTTCGT) via lentiviral transduction.
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Nakamura, Watanabe, Ando, Ishihara, and Sato. "Transplacental Gene Delivery (TPGD) as a Noninvasive Tool for Fetal Gene Manipulation in Mice." International Journal of Molecular Sciences 20, no. 23 (November 25, 2019): 5926. http://dx.doi.org/10.3390/ijms20235926.
Full textAbstract:
Transplacental gene delivery (TPGD) is a technique for delivering nucleic acids to fetal tissues via tail-vein injections in pregnant mice. After transplacental transport, administered nucleic acids enter fetal circulation and are distributed among fetal tissues. TPGD was established in 1995 by Tsukamoto et al., and its mechanisms, and potential applications have been further characterized since. Recently, discoveries of sequence specific nucleases, such as zinc-finger nuclease (ZFN), transcription activator-like effector nucleases (TALEN), and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) (CRISPR/Cas9), have revolutionized genome editing. In 2019, we demonstrated that intravenous injection of plasmid DNA containing CRISPR/Cas9 produced indels in fetal myocardial cells, which are comparatively amenable to transfection with exogenous DNA. In the future, this unique technique will allow manipulation of fetal cell functions in basic studies of fetal gene therapy. In this review, we describe developments of TPGD and discuss their applications to the manipulation of fetal cells.
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Hasanzadeh, Akbar, Fatemeh Radmanesh, Elaheh Sadat Hosseini, Iman Hashemzadeh, Jafar Kiani, Marzieh Naseri, Helena Nourizadeh, et al. "Synthesis and characterization of vitamin D3-functionalized carbon dots for CRISPR/Cas9 delivery." Nanomedicine 16, no. 19 (August 2021): 1673–90. http://dx.doi.org/10.2217/nnm-2021-0038.
Full textAbstract:
Aim: To develop a novel nanovector for the delivery of genetic fragments and CRISPR/Cas9 systems in particular. Materials & methods: Vitamin D3-functionalized carbon dots (D/CDs) fabricated using one-step microwave-aided methods were characterized by different microscopic and spectroscopic techniques. The 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl tetrazolium bromide assay and flow cytometry were employed to determine the cell viability and transfection efficiency. Results: D/CDs transfected CRISPR plasmid in various cell lines with high efficiency while maintaining their remarkable efficacy at high serum concentration and low plasmid doses. They also showed great potential for the green fluorescent protein disruption by delivering two different types of CRISPR/Cas9 systems. Conclusion: Given their high efficiency and safety, D/CDs provide a versatile gene-delivery vector for clinical applications.
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Wang, Yanchi, Junya Zhao, Nannan Duan, Wei Liu, Yuxuan Zhang, Miaojin Zhou, Zhiqing Hu, et al. "Paired CRISPR/Cas9 Nickases Mediate Efficient Site-Specific Integration of F9 into rDNA Locus of Mouse ESCs." International Journal of Molecular Sciences 19, no. 10 (October 5, 2018): 3035. http://dx.doi.org/10.3390/ijms19103035.
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Hemophilia B (HB) is an X-linked recessive bleeding disorder, caused by F9 gene deficiency. Gene therapy combined with the CRISPR/Cas9 technology offers a potential cure for hemophilia B. Now the Cas9 nickase (Cas9n) shows a great advantage in reducing off-target effect compared with wild-type Cas9. In this study, we found that in the multicopy ribosomal DNA (rDNA) locus, the homology directed recombination (HDR) efficiency induced by sgRNA-Cas9n was much higher than sgRNA-Cas9, meanwhile without off-target in six predicted sites. After co-transfection into mESCs with sgRNA-Cas9n and a non-viral rDNA targeting vector pMrnF9, harboring the homology donor template and the human F9 expression cassette, a recombination efficiency of 66.7% was achieved and all targeted clones were confirmed to be site-specific integration of F9 in the rDNA locus by PCR and southern blotting. Targeted mESCs retained the main pluripotent properties and were then differentiated into hepatic progenitor like cells (HPLCs) and mature hepatocytes, which were characterized by hepatic markers and functional assays. Importantly, the differentiated cells could transcribe exogenous F9 and secrete coagulation factor IX (FIX) proteins, suggesting active transcription and stable inheritance of transgenes in the rDNA locus. After intrasplenical transplantation in severe combined immune deficiency (SCID) mice, targeted HPLCs could survive and migrate from spleen to liver, resulting in secretion of exogenous FIX into blood. In summary, we demonstrate an efficient and site-specific gene targeting strategy in rDNA locus for stem cell-based gene therapy for hemophilia B.
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Chang, Sean, Nektaria Andronikou, and Xavier de Mollerat du Jeu. "Optimization of Gene Editing in Human Primary T Cells with Neon® Transfection System." Journal of Immunology 198, no. 1_Supplement (May 1, 2017): 73.24. http://dx.doi.org/10.4049/jimmunol.198.supp.73.24.
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Abstract The successful cases of autologous CAR-T cell therapy in leukemia have highlighted the promising future of cell therapy. However, most studies in this field have been using viruses to engineer T cells, which lead to safety concerns. On the other hand, there has been a growing interest in developing allogeneic immune cell therapies to tackle the challenges of production scale up. Hence, there is a need for a better delivery system to maximize gene editing efficiency in immune cells. Neon® Transfection System has been widely used in a variety of cell models and especially hard-to-transfect cells, such as primary cells and stem cells. Here we demonstrate how Neon® is fully capable of supporting all spectrums of research in cell therapy, including RNA delivery, DNA delivery, and CRISPR/Cas9 gene editing. Human primary T cells from four different donors were isolated from individual fresh Leukapheresis Pak, followed by activation/expansion in OpTmizer media with 2% human serum and CD3/CD28 Dynabeads. The optimal delivery time was between 3–5 days after activation. The transfection efficiency can be reached over 95% for RNA (EGFP mRNA) and 85% for DNA (pcDNAEF1a-emGFP) with high viability using certain Neon® programs. For CRISPR/Cas9 gene editing, more than 30% knock-in efficiency was observed while cleavage efficiency at HPRT locus can be reached more than 70%. The promising results of gene delivery and editing in primary T cells from Neon® could facilitate the progress of immunotherapy. Meanwhile, we have been dedicating in developing a next generation cGMP-certified large-scale electroporation system, which enable researchers to seamlessly scale-up from Neon®.
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Zhao, Xiao-Yong, and Xiao-Li Zhang. "DNA Methyltransferase Inhibitor 5-AZA-DC Regulates TGFβ1-Mediated Alteration of Neuroglial Cell Functions after Oxidative Stress." Oxidative Medicine and Cellular Longevity 2022 (September 28, 2022): 1–13. http://dx.doi.org/10.1155/2022/9259465.
Full textAbstract:
5-AZA-DC is an efficient methylation inhibitor that inhibits methylation of target DNA. In this study, we explored the effects of 5-AZA-DC on the regulation of TGFβ1 on target genes in neuroglial cell, as well as neuroglial cell functions under oxidative stress. The oxidative stress was constructed by editing CRISPR/Cas9 for knock out Ang-1 and ApoE4 genes. Cells were subjected to TGFβ1OE (or shTGFβ1) transfection and/or 5-AZA-DC intervention. Results showed that under oxidative stress, both TGFβ1OE and shTGFβ1 transfection raised DNMT1, but reduced TGFβ1, PTEN, and TSC2 expressions in neuroglial cells. TGFβ1 directly bind to the promoter of PTEN gene. 5-AZA-DC intervention lowered DNMT1 and raised TGFβ1 expression, as well as promoted the binding between TGFβ1 and promoter of PTEN. TGFβ1OE caused a significant increase in the DNA demethylation level of PTEN promoter, while 5-AZA-DC intervention reduced the DNA demethylation level of PTEN promoter. Under oxidative stress, TGFβ1OE (or shTGFβ1) transfection inhibited neuroglial cell proliferation, migration, and invasion, promoted cell apoptosis. 5-AZA-DC intervention alleviated TGFβ1OE (or shTGFβ1) transfection caused neuroglial cell proliferation, migration, and invasion inhibition, as well as cell apoptosis. To conclude, these results suggest that 5-AZA-DC can be used as a potential drug for epigenetic therapy on oxidative stress damage in neuroglial cells. The findings of this research provide theoretical basis and research ideas for methylation drug intervention and TGFβ1 gene as a possible precise target of glial oxidative stress diagnosis and treatment.
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Krachulec, Justyna M., Georg Sedlmeier, Wilko Thiele, and Jonathan P. Sleeman. "Footprintless disruption of prosurvival genes in aneuploid cancer cells using CRISPR/Cas9 technology." Biochemistry and Cell Biology 94, no. 3 (June 2016): 289–96. http://dx.doi.org/10.1139/bcb-2015-0150.
Full textAbstract:
CRISPR/Cas9 has emerged as a powerful methodology for the targeted editing of genomic DNA sequences. Nevertheless, the intrinsic inefficiency of transfection methods required to use this technique with cultured cells requires the selection and isolation of successfully modified cells, which invariably subjects the cells to stress. Here we report a workflow that allows the isolation of genomically modified cells, even where loss of functional alleles constitutes a selective disadvantage owing to impaired ability to survive stress. Using targeted disruption of the Id1 and Id3 genes in murine B16-F10 and Ret melanoma cell lines as an example, we show that the method allows for the footprintless isolation of CRISPR/Cas9-modified aneuploid cancer cells. We also provide evidence that serial CRISPR/Cas9 modifications can occur, for example when initial homologous recombination events introduce cryptic PAM sequences, and demonstrate that multiple alleles can be successfully targeted in aneuploid cancer cells. By sequencing individual alleles we also found evidence for CRISPR/Cas9-induced transposable element insertion, albeit at a low frequency. This workflow should have broad application in the functional analysis of prosurvival gene function in cultured cells.
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Chang, Sean, Xin Yu, Yongchang Ji, Xiquan Liang, Nektaria Andronikou, and Xavier de Mollerat du Jeu. "Non-viral and viral delivery solutions for next generation cell therapy." Journal of Immunology 200, no. 1_Supplement (May 1, 2018): 179.14. http://dx.doi.org/10.4049/jimmunol.200.supp.179.14.
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Abstract The successes of chimeric antigen receptor (CAR) T cells in treating blood cancers have highlighted the cell therapy era. However, the difficulty of delivering molecules into immune cells has been an obstacle to more rapid advancement. Here we present an innovative large-scale Lentivirus (LV) production system as a solution to lower the cost and time of viral production. On the other hand, the next generation cell therapy will rely heavily on gene editing, especially in a safer non-viral integration manner. We have demonstrated that our novel non-viral all-in-one electroporation method provides high efficiency of gene knock-in in primary T cells. The new LV production system was developed for the clinical grade production of lentiviral vectors (LVVs) on a large-scale serum-free suspension platform. This technology employs a newly developed propriety set of GMP reagents comprising of culture media, suspension cells, transfection reagent and boosting enhancers. The system is able to deliver greater than 1 × 108 (TU/mL) functional titer with un-concentrated LVVs. For CRISPR/Cas9 gene editing in primary T cells, we were able to reach more than 90% knockout efficiency for most genes we tested, including T cell receptor (TCR), with Cas9 RNP electroporation using Neon Transfection System. More importantly, gene knock-in efficiency can be reached to greater than 30% with all-in-one electroporation, which delivers Cas9 RNP and donor DNA in one reaction using our newly developed electroporation buffer. Moreover, TCR knockout in addition to a knock-in at another locus can also be done in a single electroporation using our all-in-one method, which is ideal for developing next generation CAR and TCR-T cells.
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Michalski, Krzysztof, Paulina Ziąbska, Sławomir Sowa, Janusz Zimny, and Anna M. Linkiewicz. "Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures." International Journal of Molecular Sciences 24, no. 3 (January 21, 2023): 2162. http://dx.doi.org/10.3390/ijms24032162.
Full textAbstract:
Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8′-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.
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Perez, Joshua, Javier Fierro, Rocio Aguilar, and Huanyu Dou. "OTME-18. Targeted CRISPR/Cas9 gene-editing regulates the brain tumor environment." Neuro-Oncology Advances 3, Supplement_2 (July 1, 2021): ii17. http://dx.doi.org/10.1093/noajnl/vdab070.069.
Full textAbstract:
Abstract Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.