Journal articles on the topic 'CRISPR/Cas9 transfection'
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Zhang, Zhen, Lei Xiong, Chao Xie, Lingling Shen, Xuanhao Chen, Min Ye, Linyang Sun, et al. "Optimization and Application of CRISPR/Cas9 Genome Editing in a Cosmopolitan Pest, Diamondback Moth." International Journal of Molecular Sciences 23, no. 21 (October 27, 2022): 13042. http://dx.doi.org/10.3390/ijms232113042.
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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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Akbaba, Hasan, Gulsah Erel-Akbaba, and Serif Senturk. "Special Focus Issue Part II: Recruitment of solid lipid nanoparticles for the delivery of CRISPR/Cas9: primary evaluation of anticancer gene editing." Nanomedicine 16, no. 12 (May 2021): 963–78. http://dx.doi.org/10.2217/nnm-2020-0412.
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Aim: The CRISPR/Cas9 system is a promising gene-editing tool for various anticancer therapies; however, development of a biocompatible, nonviral and efficient delivery of CRISPR/Cas9 expression systems remains a challenge. Materials & methods: Solid lipid nanoparticles (SLNs) were produced based on pseudo and 3D ternary plots. Obtained SLNs and their complexes with PX458 plasmid DNA were characterized and evaluated in terms of cytotoxicity and transfection efficiency. Results: SLNs were found to be nanosized, monodispersed, stable and nontoxic. Furthermore, they revealed similar transfection efficiency as the positive control. Conclusion: Overall, we have achieved a good SLN basis for CRISPR/Cas9 delivery and have the potential to produce SLNs with targeted anticancer properties by modifying production parameters and components to facilitate translating CRISPR/Cas9 into preclinical studies.
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Lanjewar, S. N., and K. R. Bondioli. "205 Optimization of Transfection Efficiency for CRISPR/Cas9-Induced Genomic Editing in Porcine Fibroblast Cells." Reproduction, Fertility and Development 30, no. 1 (2018): 243. http://dx.doi.org/10.1071/rdv30n1ab205.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas9) system creates DNA double-stranded breaks (DSB) at specific sequences and allows efficient genomic modification, even in species previously resistant to gene editing. The DSB can be repaired using non-homologous end joining (creating insertions/deletions) or by homology directed repair (HDR) using a donor DNA with small changes at the cut site, giving rise to precise targeted modifications. Despite growing interest in genome editing using RNA-guided endonucleases, the efficiency of HDR is only 0.5 to 20%. The objective of this study was to optimize transfection conditions in order to increase efficiency of HDR for CRISPR/Cas9 targeted genomic editing of porcine cells. We utilised the Swedish mutation of the porcine APP gene causing early-onset Alzheimer’s disease. We first tested co-transfection of 2 plasmids, one containing our guide RNA (gRNA) and another containing the Cas9 nuclease, using square-wave electroporation. Upon analysis via T7 endonuclease assay I, this method failed to produce a DNA DSB at the target site. Next, we tested transfection of a single vector containing both the gRNA and Cas9 nuclease. Three gRNAs targeting exon 17 of the porcine APP gene were constructed and inserted into CRISPR/Cas9 pGuide-it plasmids expressing green fluorescent protein (GFP). Plasmid DNA was transfected into cultured porcine fibroblast cells by 3 methods: Lipofectamine 2000, square-wave electroporation, and exponential-wave electroporation. To determine which method yielded the highest transfection rates, cells were analysed using flow cytometry to detect GFP expression. The transfection efficiency, percentage of cells expressing GFP, was analysed by one-way ANOVA and individual pair wise comparisons. Twelve microliters of Lipofectamine 2000 per well of a 6-well plate with 200 ng of plasmid DNA per μL of Lipofectamine was used to optimize transfection rates, as suggested by the manufacturer. Removal of transfection media after 48 h yielded higher transfection rates than removal after 24 h (6.9% ± 0.7 v. 2.2% ± 0.1; P = 0.02). For electroporation, 12.5 and 25 μg of plasmid DNA per mL was added to 0.2- and 0.4-mm gap cuvettes, respectively, each containing cell suspensions of 1 × 106 cells mL−1. Square-wave electroporation was performed at 300 V for three 1-ms pulses in 0.2-mm cuvettes. Exponential-wave electroporation was performed at 350 V and 500 μFD in both 0.2-mm and 0.4-mm cuvettes. Exponential-wave electroporation containing 25 μg of plasmid DNA/mL of cell suspension yielded the highest average transfection efficiency, 22.8% (P < 0.00001), compared with square-wave electroporation and transfection using optimized Lipofectamine 2000 conditions (9.1 and 1%, respectively). All 3 gRNAs resulted in similar transfection rates. In conclusion, efficiency of transfection of the CRISPR/Cas9 system into porcine cells is optimized using exponential-wave electroporation of a single plasmid CRISPR system.
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Nasri, Masoud, Perihan Mir, Benjamin Dannenmann, Diana Amend, Yun Xu, Anna Solovyeva, Sylwia Stefanczyk, et al. "A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs." Blood 132, Supplement 1 (November 29, 2018): 1108. http://dx.doi.org/10.1182/blood-2018-99-114844.
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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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Moradi, Pardis, Akbar Hasanzadeh, Fatemeh Radmanesh, Saideh Rajai Daryasarei, Elaheh Sadat Hosseini, Jafar Kiani, Ali Shahbazi, et al. "Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells." Nanotechnology 33, no. 7 (November 26, 2021): 075104. http://dx.doi.org/10.1088/1361-6528/ac357a.
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Abstract An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8k in the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5–10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid and in vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.
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Cheng, Hao, Feng Zhang, and Yang Ding. "CRISPR/Cas9 Delivery System Engineering for Genome Editing in Therapeutic Applications." Pharmaceutics 13, no. 10 (October 9, 2021): 1649. http://dx.doi.org/10.3390/pharmaceutics13101649.
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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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McAndrews, Kathleen M., Fei Xiao, Antonios Chronopoulos, Valerie S. LeBleu, Fernanda G. Kugeratski, and Raghu Kalluri. "Exosome-mediated delivery of CRISPR/Cas9 for targeting of oncogenic KrasG12D in pancreatic cancer." Life Science Alliance 4, no. 9 (July 19, 2021): e202000875. http://dx.doi.org/10.26508/lsa.202000875.
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CRISPR/Cas9 is a promising technology for gene editing. To date, intracellular delivery vehicles for CRISPR/Cas9 are limited by issues of immunogenicity, restricted packaging capacity, and low tolerance. Here, we report an alternative, nonviral delivery system for CRISPR/Cas9 based on engineered exosomes. We show that non-autologous exosomes can encapsulate CRISPR/Cas9 plasmid DNA via commonly available transfection reagents and can be delivered to recipient cancer cells to induce targeted gene deletion. As a proof-of-principle, we demonstrate that exosomes loaded with CRISPR/Cas9 can target the mutant KrasG12D oncogenic allele in pancreatic cancer cells to suppress proliferation and inhibit tumor growth in syngeneic subcutaneous and orthotopic models of pancreatic cancer. Exosomes may thus be a promising delivery platform for CRISPR/Cas9 gene editing for targeted therapies.
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Aschenbrenner, Sabine, Stefan M. Kallenberger, Mareike D. Hoffmann, Adrian Huck, Roland Eils, and Dominik Niopek. "Coupling Cas9 to artificial inhibitory domains enhances CRISPR-Cas9 target specificity." Science Advances 6, no. 6 (February 2020): eaay0187. http://dx.doi.org/10.1126/sciadv.aay0187.
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The limited target specificity of CRISPR-Cas nucleases poses a challenge with respect to their application in research and therapy. Here, we present a simple and original strategy to enhance the specificity of CRISPR-Cas9 genome editing by coupling Cas9 to artificial inhibitory domains. Applying a combination of mathematical modeling and experiments, we first determined how CRISPR-Cas9 activity profiles relate to Cas9 specificity. We then used artificially weakened anti-CRISPR (Acr) proteins either coexpressed with or directly fused to Cas9 to fine-tune its activity toward selected levels, thereby achieving an effective kinetic insulation of ON- and OFF-target editing events. We demonstrate highly specific genome editing in mammalian cells using diverse single-guide RNAs prone to potent OFF-targeting. Last, we show that our strategy is compatible with different modes of delivery, including transient transfection and adeno-associated viral vectors. Together, we provide a highly versatile approach to reduce CRISPR-Cas OFF-target effects via kinetic insulation.
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Aguilar, Rocio, Javier Fierro, Joshua Perez, and Huanyu Dou. "OMRT-12. Nanoparticle-based CRISPR-Cas9 delivery for anti-glioblastoma immunotherapy." Neuro-Oncology Advances 3, Supplement_2 (July 1, 2021): ii9. http://dx.doi.org/10.1093/noajnl/vdab070.036.
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Abstract Anti-glioblastoma GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) is an immune checkpoint that mediated the immune resistance. Inhibition of PD-L1 by antibodies was widely studied to treat many type of cancers. However, the inefficient therapeutic immune response became a significant barrier for treatment of GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. It is extremely difficulty to deliver CRISPR/Cas9 containing plasmid for translational and clinic applications. We have been developed a core-shell nanoparticle (NP) to carry CRISPR/Cas9 plasmid for PD-L1 knockout. The different NP formulations were made and optimized to deliver CRISPR/Cas9 plasmid. NPs were prepared by modifying the water temperature, sonication power and time and formulation time. The obtained NPs had a size of 115-160nm and a charge of 40-50mV. The size and charge were significantly altered after CRISPR/Cas9 plasmids were loaded into NPs (Cas9-NPs). Agarose gel electrophoresis showed that CRISPR/Cas9 plasmids were fully encapsulated by NPs with 1 and 2 ug. The positive DNA bands occurred with 4ng, indicating the overloaded CRISPR/Cas9 plasmid. Fluorescence microscopy determined Cas9-NPs uptake by U87 cells under a time-dependent manner. GFP tagged Cas9-NPs were treated to U87 cells for transfection evaluation. The obtained different NPs delivery of CRISPR/Cas9 exhibited various transfection efficiencies in U87 cells. Visualization of intracellular Cas9-NPs showed increases in uptake by U87 cells from 0.5, 1, 2, and 4 hours. The greater nuclear accumulation of Cas9-NPs was seen at 24 hours. A western blot assay determined the success of PD-L1 deletion by Cas9-NPs in human GBM U87 cells. NPs-based CRISPR/Cas9 gene-editing system has great potential as an immunotherapeutic platform to treat GBM.
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Atanes, Patricio, Inmaculada Ruz-Maldonado, Ross Hawkes, Bo Liu, Shanta J. Persaud, and Stefan Amisten. "Identifying Signalling Pathways Regulated by GPRC5B in β-Cells by CRISPR-Cas9-Mediated Genome Editing." Cellular Physiology and Biochemistry 45, no. 2 (2018): 656–66. http://dx.doi.org/10.1159/000487159.
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Background/Aims: CRISPR-Cas9, a RNA-guided targeted genome editing tool, has revolutionized genetic engineering by offering the ability to precisely modify DNA. GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). In this study, we analysed the functional roles of the Gprc5b receptor in MIN6 β-cells using CRISPR-Cas9 and transient over-expression of Gprc5b. Methods: The optimal transfection reagent for use in MIN6 β-cells was determined by analysing efficiency of GFP plasmid delivery by cell sorting. A MIN6 β-cell line in which Gprc5b expression was knocked down (Gprc5b KD) was generated using CRISPR-Cas9 technology. Gprc5b receptor mRNA expression, proliferation, apoptosis, Cignal 45-Pathway Reporter Array signalling and western blot assays were carried out using Gpcr5b KD MIN6 β-cells that had been transiently transfected with different concentrations of mouse Gprc5b plasmid to over-express Gprc5b. Results: JetPRIME® was the best candidate for MIN6 β-cell transfection, providing approximately 30% transfection efficiency. CRISPR-Cas9 technology targeting Gprc5b led to stable knock-down of this receptor in MIN6 β-cells and its re-expression induced proliferation and potentiated cytokine- and palmitate-induced apoptosis. The Cignal 45 Reporter analysis indicated Gprc5b-dependent regulation of apoptotic and proliferative pathways, and western blotting confirmed activation of signalling via TGF-β and IFNγ. Conclusion: This study provides evidence of CRISPR-Cas9 technology being used to down-regulate Gprc5b expression in MIN6 β-cells. This strategy allowed us to identify signalling pathways linking GPRC5B receptor expression to β-cell proliferation and apoptosis.
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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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Mohammadi Ghanbarlou, Mahdi, Shahriyar Abdoli, Hamed Omid, Leila Qazizadeh, Hadi Bamehr, Mozhgan Raigani, Hosein Shahsavarani, Morteza Karimipour, and Mohammad Ali Shokrgozar. "Delivery of dCas9 Activator System Using Magnetic Nanoparticles Technology as a Vector Delivery Method for Human Skin Fibroblast." Magnetochemistry 9, no. 3 (February 28, 2023): 71. http://dx.doi.org/10.3390/magnetochemistry9030071.
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The overexpression of stem cell-related genes such as octamer-binding transcription factor 4 (OCT4) and (sex determining region Y)-box 2 (SOX2) has been indicated to play several critical roles in stem cell self-renewal; moreover, the elevation of the self-renewal of cancer cells with stem cell-like properties has been suggested. The clustered and regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) protein fused to transactivation domains can be used to activate gene expression in human cells. CRISPR-mediated activation (CRISPRa) systems represent an effective genome editing tool for highly specific gene activation in which a nuclease-deficient Cas9 (dCas9) is utilized to target a transcriptional activator to the gene’s regulatory element, such as a promoter and enhancer. The main drawback of typical delivery methods for CRISPR/Cas9 components is their low transfection efficiency or toxic effects on cells; thus, we generated superparamagnetic iron oxide nanoparticles (SPIONs) coated with polyethylenimine (PEI) to improve the delivery of CRISPR/Cas9 constructs into human foreskin fibroblast cells. The delivery system with magnetic PEI-coated nanoparticles complex was applied to constitute plasmid DNA lipoplexes. CRISPRa systems were used to overexpress the endogenous OCT4 and SOX2 in fibroblast cells. The quantitative polymerase chain reaction (QPCR) assessment exhibited a three-times higher expression of OCT4 and SOX2 transfected by CRISPRa using MNPs. Moreover, no additional cytotoxicity was observed with the application of magnetic nanoparticles (MNPs) compared to lipofectamine. Our results demonstrate that MNPs enable the effective delivery of the CRISPR/Cas9 construct into human foreskin fibroblasts with low cell toxicity and a consequential overexpression of endogenous OCT4 and SOX2.
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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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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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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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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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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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Chen, Li, Kaikai Shi, Weimin Qiu, Lars Aagaard, and Moustapha Kassem. "Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation." Stem Cells International 2020 (August 19, 2020): 1–11. http://dx.doi.org/10.1155/2020/8857344.
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Background. Human bone marrow stromal/stem cells (hMSCs, also known as the skeletal stem cells or mesenchymal stem cells) are being employed to study lineage fate determination to osteoblasts, adipocytes, and chondrocytes. However, mechanistic studies employing hMSC have been hampered by the difficulty of deriving genetically modified cell lines due to the low and unstable transfection efficiency. Methods. We infected hMSC with a CRISPR/Cas9 lentivirus system, with specific inducible dCas9-coupled transcription activator or repressor: dCas9-KRAB or dCas9-VP64, respectively, and established two hMSC lines (hMSC-CRISPRi and hMSC-CRISPRa) that can inhibit or activate gene expression, respectively. The two cell lines showed similar cell morphology, cell growth kinetics, and similar lineage differentiation potentials as the parental hMSC line. The expression of KRAB-dCas9 or VP64-dCas9 was controlled by the presence or absence of doxycycline (Dox) in the cell culturing medium. To demonstrate the functionality of the dCas9-effector hMSC system, we tested controlled expression of alkaline phosphatase (ALP) gene through transfection with the same single ALP sgRNA. Results. In the presence of Dox, the expression of ALP showed 60-90% inhibition in hMSC-CRISPRi while ALP showed more than 20-fold increased expression in hMSC-CRISPRa. As expected, the ALP was functionally active and the cells showed evidence for inhibition or enhancement of in vitro osteoblast differentiation, respectively. Conclusion. hMSC-CRISPRi and hMSC-CRISPRa are useful resources to study genes and genetic pathways regulating lineage-specific differentiation of hMSC.
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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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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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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.
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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.
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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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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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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.
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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.
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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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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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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.
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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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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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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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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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Wardhani, Bantari W. K., Meidi U. Puteri, Yukihide Watanabe, Melva Louisa, Rianto Setiabudy, and Mitsuyasu Kato. "TMEPAI genome editing in triple negative breast cancer cells." Medical Journal of Indonesia 26, no. 1 (May 16, 2017): 14–8. http://dx.doi.org/10.13181/mji.v26i1.1871.
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Background: Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) is a powerful genome editing technique. It consists of RNA-guided DNA endonuclease Cas9 and single guide RNA (gRNA). By combining their expressions, high efficiency cleavage of the target gene can be achieved, leading to the formation of DNA double-strand break (DSB) at the genomic locus of interest which will be repaired via NHEJ (non-homologous end joining) or HDR (homology-directed repair) and mediate DNA alteration. We aimed to apply the CRISPR/Cas9 technique to knock-out the transmembrane prostate androgen-induced protein (TMEPAI) gene in the triple negative breast cancer cell line.Methods: Designed gRNA which targets the TMEPAI gene was synthesized, annealed, and cloned into gRNA expression vector. It was co-transfected into the TNBC cell line using polyethylenimine (PEI) together with Cas9-GFP and puromycin resistant gene vector. At 24-hours post-transfection, cells were selected by puromycin for 3 days before they were cloned. Selected knock-out clones were subsequently checked on their protein levels by western blotting.Results: CRISPR/Cas9, a genome engineering technique successfully knocked-out TMEPAI in the Hs578T TNBC cell line. Sequencing shows a frameshift mutation in TMEPAI. Western blot shows the absence of TMEPAI band on Hs578T KO cells.Conclusion: TMEPAI gene was deleted in the TNBC cell line using the genomic editing technique CRISPR/Cas9. The deletion was confirmed by genome and protein analysis.
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Kostyusheva, A. P., S. A. Brezgin, D. N. Zarifyan, D. S. Chistyakov, V. I. Gegechkory, E. O. Bayurova, E. A. Volchkova, D. S. Kostyushev, and V. P. Chulanov. "Hepatitis B virus and site-specific nucleases: effects of genetic modifications in CRISPR/Cas9 on antiviral activity." Russian Journal of Infection and Immunity 9, no. 2 (July 12, 2019): 279–87. http://dx.doi.org/10.15789/2220-7619-2019-2-279-287.
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Chronic hepatitis B is a severe liver disease caused by persistent infection of hepatitis B virus in human hepatocytes. Chronic hepatitis B is one of the most common diseases in the world. According to recent estimations, more than 250 million people are chronically infected and more than 1 million of people die annually due to consequences of chronic hepatitis B: liver cirrhosis and hepatocellular carcinoma. The key factor of hepatitis B virus persistency is a special form of viral genome called circular covalently closed DNA. Current therapeutics suppress viral replication but have no effect on circular covalently closed DNA as it exists in the nuclei of hepatocytes as a minichromosome and is not accessible for therapeutics. Commonly, viral reactivation occurs after cessation of treatment. Therefore, duration of antiviral treatment is supposed to be indefinitely long. One of the most promising approaches to target circular covalently closed DNA is the technology of site-specific nucleases CRISPR/Cas9 from Streptococcus pyogenes. A short guide RNA recruits an SpCas9 protein to the viral genome and induces generation of DNA double strand breaks. However, there are several limitations of CRISPR/Cas9 hampering translation of this technology into the clinic. First, efficacy of CRISPR/Cas9 needs to be improved. Second, CRISPR/Cas9-mediated off-target mutagenesis represents a menacing problem which has to be addressed. To overcome these limitations, several approaches have been devised to improve CRISPR/Cas9 activity (modification of guide RNAs) and reduce off-target mutagenesis (a Cas9 protein with enhanced specificity, eSpCas9). In this study, we compared antiviral activity of a classic SpCas9 with an eSpCas9 system as well as analyzed effects of gRNAs modification on anti-HBV effects. Here, we demonstrated that SpCas9 has the highest antiviral potency, reducing transcription and replication of HBV over 90%. Hepatitis B virus covalently closed circular DNA declined over 90% post CRISPR/Cas9 transfection. Although it was previously shown that modified guide RNAs increase nucleolytic activity of CRISPR/Cas9, our results indicated that this modification impairs antiviral activity of CRISPR/Cas9. To conclude, CRISPR/Cas9 effectively suppress viral replication and transcription per se. Described modifications do not potentiate antiviral activity of CRISPR/Cas9 system and should not be used for development of future therapeutics. The best strategy to improve CRISPR/Cas9 efficacy is to design new highly effective guide RNAs.
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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.
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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.
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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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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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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.
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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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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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Bernard, Guillaume, David Gagneul, Harmony Alves Dos Santos, Audrey Etienne, Jean-Louis Hilbert, and Caroline Rambaud. "Efficient Genome Editing Using CRISPR/Cas9 Technology in Chicory." International Journal of Molecular Sciences 20, no. 5 (March 6, 2019): 1155. http://dx.doi.org/10.3390/ijms20051155.
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CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated with protein CAS9) is a genome-editing tool that has been extensively used in the last five years because of its novelty, affordability, and feasibility. This technology has been developed in many plant species for gene function analysis and crop improvement but has never been used in chicory (Cichorium intybus L.). In this study, we successfully applied CRISPR/Cas9-mediated targeted mutagenesis to chicory using Agrobacterium rhizogenes-mediated transformation and protoplast transfection methods. A U6 promoter (CiU6-1p) among eight predicted U6 promoters in chicory was selected to drive sgRNA expression. A binary vector designed to induce targeted mutations in the fifth exon of the chicory phytoene desaturase gene (CiPDS) was then constructed and used to transform chicory. The mutation frequency was 4.5% with the protoplast transient expression system and 31.25% with A. rhizogenes-mediated stable transformation. Biallelic mutations were detected in all the mutant plants. The use of A. rhizogenes-mediated transformation seems preferable as the regeneration of plants is faster and the mutation frequency was shown to be higher. With both transformation methods, foreign DNA was integrated in the plant genome. Hence, selection of vector (transgene)-free segregants is required. Our results showed that genome editing with CRISPR/Cas9 system can be efficiently used with chicory, which should facilitate and accelerate genetic improvement and functional biology.
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Chen, Xiaohong, Yuxuan Chen, Huhu Xin, Tao Wan, and Yuan Ping. "Near-infrared optogenetic engineering of photothermal nanoCRISPR for programmable genome editing." Proceedings of the National Academy of Sciences 117, no. 5 (January 15, 2020): 2395–405. http://dx.doi.org/10.1073/pnas.1912220117.
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We herein report an optogenetically activatable CRISPR-Cas9 nanosystem for programmable genome editing in the second near-infrared (NIR-II) optical window. The nanosystem, termed nanoCRISPR, is composed of a cationic polymer-coated Au nanorod (APC) and Cas9 plasmid driven by a heat-inducible promoter. The APC not only serves as a carrier for intracellular plasmid delivery but also can harvest external NIR-II photonic energy and convert it into local heat to induce the gene expression of the Cas9 endonuclease. Due to high transfection activity, the APC shows strong ability to induce a significant level of disruption in different genomic loci upon optogenetic activation. Moreover, the precise control of genome-editing activity can be simply programmed by finely tuning exposure time and irradiation time in vitro and in vivo and also enables editing at multiple time points, thus proving the sensitivity and inducibility of such an editing modality. The NIR-II optical feature of nanoCRISPR enables therapeutic genome editing at deep tissue, by which treatment of deep tumor and rescue of fulminant hepatic failure are demonstrated as proof-of-concept therapeutic examples. Importantly, this modality of optogenetic genome editing can significantly minimize the off-target effect of CRISPR-Cas9 in most potential off-target sites. The optogenetically activatable CRISPR-Cas9 nanosystem we have developed offers a useful tool to expand the current applications of CRISPR-Cas9, and also defines a programmable genome-editing strategy toward high precision and spatial specificity.
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Di Stazio, Mariateresa, Nicola Foschi, Emmanouil Athanasakis, Paolo Gasparini, and Adamo Pio d’Adamo. "Systematic analysis of factors that improve homologous direct repair (HDR) efficiency in CRISPR/Cas9 technique." PLOS ONE 16, no. 3 (March 5, 2021): e0247603. http://dx.doi.org/10.1371/journal.pone.0247603.
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The CRISPR/Cas9 bacterial system has proven to be an powerful tool for genetic manipulation in several organisms, but the efficiency of sequence replacement by homologous direct repair (HDR) is substantially lower than random indel creation. Many studies focused on improving HDR efficiency using double sgRNA, cell synchronization cycle, and the delivery of single-stranded oligo DNA nucleotides (ssODN) with a rational design. In this study, we evaluate these three methods’ synergistic effects to improve HDR efficiency. For our tests, we have chosen the TNFα gene (NM_000594) for its crucial role in various biological processes and diseases. For the first time, our results showed how the use of two sgRNA with asymmetric donor design and triple transfection events dramatically increase the HDR efficiency from an undetectable HDR event to 39% of HDR efficiency and provide a new strategy to facilitate CRISPR/Cas9-mediated human genome editing. Besides, we demonstrated that the TNFα locus could be edited with CRISPR/Cas9 methodology, an opportunity to safely correct, in the future, the specific mutations of each patient.
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Kolb, Alexander L., Marinaliz Reynoso, and Ronald W. Matheny. "Comparison of CRISPR and adenovirus-mediated Myd88 knockdown in RAW 264.7 cells and responses to lipopolysaccharide stimulation." Journal of Biological Methods 8, no. 3 (July 15, 2021): e151. http://dx.doi.org/10.14440/jbm.2021.359.
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Genomic manipulation offers the possibility for novel therapies in lieu of medical interventions in use today. The ability togenetically restore missing inflammatory genes will have a monumental impact on our current immunotherapy treatments. This study compared the efficacy of two different genetic manipulation techniques: clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) transfection to adenoviral transduction to determine which method would provide the most transient and stable knockdown of myeloid differentiation primary response 88 (MyD88). MyD88 is a major regulator of nuclear factor kappa light chain enhancer of activated B cells (NFκB) pathway in Raw 264.7 macrophages. Following genetic manipulation, cells were treated for 24 h with Lipopolysaccharide (LPS) to stimulate the inflammatory pathway. Confirmation of knockdown was determined by western immunoblotting and quantification of band density. Both CRISPR/Cas9 and adenoviral transduction produced similar knockdown efficiency (~64% and 60%, respectively) in MyD88 protein 48 h post adenoviral transduction. NFκB phosphorylation was increased in CRISPR/Cas9-mediated MyD88 knockdown and control cells, but not in adenovirus-mediated MyD88 knockdown cells, following LPS administration. CRISPR/Cas9-mediated MyD88 knockdown macrophages treated with LPS for 24 h showed a 65% reduction in tumor necrosis factor alpha (TNFα) secretion, and a 67% reduction in interleukin-10 (IL-10) secretion when compared to LPS-stimulated control cells (P ≤ 0.01 for both). LPS did not stimulate TNFα or IL-10 secretion in adenovirus-mediated control or MyD88 knockdown cells. These data demonstrate that Raw 264.7 macrophages maintain responsiveness to inflammatory stimuli following CRISPR/Cas9-mediated reductions in MyD88, but not following adenovirus-mediated MyD88 knockdown.
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Inwood, Sarah L., Linhua Tian, Samantha Maragh, and Lili Wang. "Analysis of stability CRISPR/Cas9 engineered B cells using flow cytometry." Journal of Immunology 204, no. 1_Supplement (May 1, 2020): 91.32. http://dx.doi.org/10.4049/jimmunol.204.supp.91.32.
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Abstract Objective CRISPR/Cas9, a widely used genome editing system, has huge potential and is being used to improve protein, cell and gene therapies. Though an exciting tool, there is not a lot of long-term data about the genomic and phenotypic stability and off-target effects that may arise during the editing process. Since the cells are used in the patient with cell and gene therapies, cell characterization is essential for safety. In support of production of safe and effective CRISPR/Cas9 engineered therapies, the objective of this work is to study both on- and off-target effects of CRISPR/Cas9 using flow cytometry on a B-lymphoblast cell line, GM24385, whose genome sequence has been well characterized. Methods The cultured B cells were characterized with a B cell panel using flow cytometry. The CD19 marker was knocked out using CRISPR/Cas9 RNPs with a nucleofection method using four gRNAs. The resulting pools were then evaluated with sequencing and a flow cytometry B cell panel and sorted using a FACSAria II flow sorter. Results The CD19− B cells were created from nucleofection of cultured GM24385 cells with CRISPR/Cas9 RNPs. The characteristics obtained from a B cell subtyping panel showed different efficiencies of CD19 knockout and different B cell subtypes. The growth rate of CD19− B cells was unpredictable over time post transfection. Additionally, different gRNAs against CD19 resulted in different editing efficiencies evaluated at genomic and proteomic levels. Conclusions Using CRISPR/Cas9 with different gRNAs to knock out CD19 in B lymphoblasts, GM24385, influences the B cell subtype profile. The work with well sequenced GM24385 enables the identification of off-target effects of the editing method, the next step of the study.
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Hu, S., M. Yang, and I. Polejaeva. "360 DOUBLE KNOCKOUT OF GOAT MYOSTATIN AND PRION PROTEIN GENE USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEAT (CRISPR)/Cas9 SYSTEMS." Reproduction, Fertility and Development 27, no. 1 (2015): 268. http://dx.doi.org/10.1071/rdv27n1ab360.
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Myostatin (MSTN) acts as a negative regulator of skeletal muscle development and growth. Inhibition of MSTN expression may be applied to enhance animal growth performance in livestock production. Prion protein (PrPc) is associated directly with the pathogenesis of the transmissible spongiform encephalopathies occurring in variety of species including human, cattle, sheep, goats and deer. Prion protein-deficient livestock may be a useful model for prion research and producing animal conferring potential disease resistance. The goal of this study was to generate MSTN/PrPc double knockout goat by using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system. We generated 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc genes, respectively. The CRISPR/Cas9 plasmids targeting each gene were respectively transfected into goat fibroblasts, and the efficiency of gene modification was determined at Day 3 using restriction fragment length polymorphism (RFLP) assay. The RFLP assay showed that CRISPR/Cas9 plasmids targeting MSTN and PrPc induced precise gene mutations with efficiency of 59 and 70%, respectively. Single cell-derived colonies were further isolated by limiting dilution after co-transfection of 2 CRISPR/Cas9 plasmids targeting MSTN and PrPc. The RFLP assay and DNA sequence analysis indicated that 9 out of 45 colonies (20%) carried simultaneous disruption of both target genes. Moreover, 5 of 9 mutant colonies (55%) had mutations in all 4 alleles of 2 genes. These double-gene knockout fibroblast cells will be used as nuclear donors for developing double knockout goat deficient in MSTN and PrPc. The CRISPR/Cas9 system represents a highly effective and facile platform for multiplex editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.This work was supported by the Utah Science Technology and Research Initiative and Utah Agricultural Experiment Station project #31294.
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Rouatbi, Nadia, Yau Mun Lim, Vivien Grant, Pedro Miguel Costa, Steven M. Pollard, Julie Tzu-Wen Wang, and Khuloud T. Al-Jamal. "CRISPR/Cas9 gene editing of brain cancer stem cells using lipid-based nano-delivery." Neuro-Oncology 21, Supplement_4 (October 2019): iv7. http://dx.doi.org/10.1093/neuonc/noz167.029.
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Abstract Despite advances in cancer therapy glioblastoma (GBM) remains one of the deadliest brain tumours. Effective therapy is restricted by the presence of multiple resistance mechanisms. Physical barriers such as the blood-brain barrier limit the brain delivery of therapeutic compounds. In addition, the presence of a subset of GBM-stem-like cells (GSCs), characterized by radio/chemoresistance, and the intratumor heterogeneity impede standard therapies from being effective. New therapeutic approaches are urgently needed. Given its high specificity, CRISPR/Cas9-mediated genome editing provides new prospects for novel therapeutic targets. While promising, in vivo application of CRISPR/Cas9 is currently hampered by poor pharmacokinetics and limited ability to cross biological membranes. The present research is designed to utilise stable nucleic acid lipid nanoparticles (SNALPs) for in vivo delivery of CRISPR/Cas9 to GSC by disrupting the epidermal growth factor receptor variant III (EGFRvIII), a GBM associated mutation, responsible for tumour cell proliferation, angiogenesis and invasion. Near Infrared fluorescence labelling and live optical imaging confirmed SNALPs uptake in GSC tumours implanted intracranially in mice after intravenous injection. Higher uptake of SNALPs in tumourous tissues compared to healthy brain tissues was further confirmed by ex vivo imaging and flow cytometry. EGFRvIII-specific sgRNA has been designed and validated in GSCs using commercial transfection regents. Studies are underway to load CRISPR/Cas9 mRNA/gRNA into SNALPs to test their in vitro gene editing efficacy. Successful in vivo delivery of CRISPR/Cas9 will represent a promising approach for identifying GBM therapeutic targets in vivo which in the long-run can be applied for GBM treatment.
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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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Jia, Xiaoqing, Qi Yao, Hui Li, and Jieping Chen. "Crispra: A Promising Tool for β-Thalassemia Treatment." Blood 136, Supplement 1 (November 5, 2020): 8. http://dx.doi.org/10.1182/blood-2020-141196.
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Induction of hemoglobin γ expression is a reliable strategy to treat β-thalassemia. Gene editing using CRISPR/Cas9 technology has been widely used. However, application in vivo is limited due to the uncertainty on genomic cleavages of Cas9. In contrast, CRISPR/Cas9-based gene activation (CRISPRa) can only locate genomic locus but not interrupt sequence. Here, we use SAM system of CRISPRa to locate and activate HBG1 and HBG2, exploring the great potential of CRISPRa for β-thalassemia treatment. WWe designed 8 single-guide RNAs (sgRNAs) online and cloned into vector SAM V2, which fused dCas9 and VP64. To test the over-expression efficiency, vector containing sgRNA and MPH (fused HSF1, p65 and MS2) were transfected into 293T cell. After 72h transfection, 293T cells were collected. Q-PCR data showed that two sgRNAs were excellent on activating HBG expression with over 1000-fold increase. WTo test the activating function in hematological cell and the persistence of hemoglobin γexpression, two screened sgRNA were transfected into NB4 cells using lentivirus system. We harvested NB4 cells at different time-point (3 day, 1 week and 2 weeks), and implemented q-PCR assay. HBG expression were increased 50-hold and 1000-hold, respectively. However, the expression were reducing over time and the intrinsic mechanism is unknown. WThis study set out to increase HBG without interrupt genome using CRISPRa system. This study has found two sgRNA to activate the expression of HBG in 293T cell and NB4 cell. Further research is required to vertify the efficiency of sgRNA in hematopoietic stem cells and prolong the expression time. Figure Disclosures No relevant conflicts of interest to declare.
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Fierro, Javier, An Tran, Chris Factoriza, Brandon Chin, and Huanyu Dou. "TAMI-14. NANOPARTICLE DELIVERY OF PD-L1 CRISPR/CAS9 PLASMID DNA FOR ANTI-GLIOBLASTOMA IMMUNOTHERAPY." Neuro-Oncology 22, Supplement_2 (November 2020): ii216. http://dx.doi.org/10.1093/neuonc/noaa215.903.
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Abstract Glioblastoma multiforme (GBM) is a devastating cancer that develops from astrocytes in the brain. GBM is fast acting and kills 90% of patients within 5 years. Several immunotherapies have been developed to treat GBM, however, major challenges still persist. For example, checkpoint proteins such as programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1), are upregulated in GBM cells to evade the immune system. Targeting PD-L1 for genetic knockdown is thus a promising avenue for the treatment of GBM. However, PD-L1 protein inhibitors have been shown to cause immune overreaction and toxicity, therefore requiring new technologies. CRISPR/Cas9 gene editing has been widely used for the study and treatment of many diseases, but has not been extensively studied for the treatment of GBM. The main challenge is developing a gene delivery platform for the delivery of CRISPR/Cas9 plasmid DNA (pDNA). Many viral vectors have been used for the delivery of pDNA, but unfortunately are associated with high toxicity. Nanotechnology is emerging as a new platform for the delivery of pDNA as it shows high transfection efficiency with low cytotoxicity. We developed a cationic core-shell nanoparticle (NP) capable of carrying CRISPR/Cas9 pDNA. This plasmid contains multiple guide RNA (gRNA) expression cassettes for the knockdown of PD-L1. PDL1gRNA-CRISPR/Cas9pDNA-NPs were taken up by U87 cells within 30 minutes, and entered into the nucleus at 2 hours. The effective delivery of PDL1gRNA-CRISPR/Cas9pDNA-NPs led to the expression of PD-L1 gRNA and Cas9 enzyme, and the knockdown of PD-L1. Regulation of immune balance was determined after PD-L1 knockdown in vitro and in vivo. Our study shows the potential of NP-based PDL1gRNA-CRISPR/Cas9 delivery as an anti-GBM immunotherapy for clinical applications.
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Lentsch, Eva, Lifei Li, Susanne Pfeffer, Arif B. Ekici, Leila Taher, Christian Pilarsky, and Robert Grützmann. "CRISPR/Cas9-Mediated Knock-Out of KrasG12D Mutated Pancreatic Cancer Cell Lines." International Journal of Molecular Sciences 20, no. 22 (November 14, 2019): 5706. http://dx.doi.org/10.3390/ijms20225706.
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In 90% of pancreatic ductal adenocarcinoma cases, genetic alteration of the proto-oncogene Kras has occurred, leading to uncontrolled proliferation of cancerous cells. Targeting Kras has proven to be difficult and the battle against pancreatic cancer is ongoing. A promising approach to combat cancer was the discovery of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, which can be used to genetically modify cells. To assess the potential of a CRISPR/CRISPR-associated protein 9 (Cas9) method to eliminate Kras mutations in cells, we aimed to knock-out the c.35G>A (p.G12D) Kras mutation. Therefore, three cell lines with a heterozygous Kras mutation (the human cell lines SUIT-2 and Panc-1 and the cell line TB32047 from a KPC mouse model) were used. After transfection, puromycin selection and single-cell cloning, proteins from two negative controls and five to seven clones were isolated to verify the knock-out and to analyze changes in key signal transduction proteins. Western blots showed a specific knock-out in the KrasG12D protein, but wildtype Kras was expressed by all of the cells. Signal transduction analysis (for Erk, Akt, Stat3, AMPKα, and c-myc) revealed expression levels similar to the wildtype. The results described herein indicate that knocking-out the KrasG12D mutation by CRISPR/Cas9 is possible. Additionally, under regular growth conditions, the knock-out clones resembled wildtype cells.
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Peyravian, Noshad, Maziar Malekzadeh Kebria, Jafar Kiani, Peiman Brouki Milan, and Masoud Mozafari. "CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip." Materials 14, no. 12 (June 9, 2021): 3164. http://dx.doi.org/10.3390/ma14123164.
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Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9–12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.