Stable integration can occur randomly with plasmids, actively at random sites with help of transposases or viruses, or site-specifically when using genome editing tools like CRISPR. A.M. is a Chan Zuckerberg Biohub investigator. DEAE-dextran is a polycationic derivative of dextran (a carbohydrate polymer). Does anyone have an optimized protocol for electroporation human dermal Furthermore, with DNA-sensitive cells, cell viability is often much better when using RNA. & Kim, J. S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Nature 540, 144149 (2016). Sadelain, M., Rivire, I. Required Material pSTAT5+ cells correlated with increased IL-2R surface expression. of technical triplicates (j). A scalable strategy for high-throughput GFP tagging of endogenous human proteins. is a Parker Institute for Cancer Immunotherapy member. Cell. CD3+CD4+CD127loCD45RO+TIGIT+ Treg cells, a population highly enriched for FOXP3+ cells (Extended Data Fig. dsDNA HDR temple (5 g) was used in ce. Thus, correcting functional IL-2R expression on the surface of FOXP3+ T cells from these patients may represent a viable approach for developing an ex vivo gene therapy. 3b). Learn more about the NucleofectorTM Technology and its different fields of application. If youd like to learn more about Lipofection and other chemical-based transfection methods, download our Transfection Methods Technical Reference Guide here. Reprogramming human T cell function and specificity with non-viral genome targeting. To confirm that such a potential correction would result in some level of functional suppression, we assessed the suppressive ability of CD4+IL-2RhiCD127lo Treg cells from the c.530 and c.800 single heterozygote family members as in f. h, Dot plot summaries of Treg cell suppressive ability in cells from healthy donors (n=3 with single (top) or 12 (bottom) technical replicates), IL2RA-deficient compound heterozygotes (f, n=3 total human subjects) and IL2RA +/ c.530 or c.800 heterozygotes (g, n=4 total human subjects). Sanger sequencing confirmed that the proband was a compound heterozygote with both mutations. Some transfection methods are effective for use with cells lines, but they may not be suitable for use with primary cells (including resting cells like unstimulated T cells or neurons) or stem cells, both of which are generally harder to work with. Nature (Nature) Transfection requires the delivery of material through the cell membrane, which is negatively charged. Article Cells transfected by Proc. ), and P30 DK020595 (S.W.G. This introduces temporary pores that allow the exogenous DNA to enter the cell. Simeonov, D. R. et al. As for all transfection methods, electroporation has its advantages and disadvantages. However, as DNA constructs must make their way into the nucleus to induce gene expression, they are slower to drive protein production than mRNA (which exerts its effects as soon as it enters the cytoplasm, see below). hets 13), with loss-of-function IL2RA mutations were analysed by flow cytometry to assess the presence of IL-2RhiCD127lo Treg cells. Methods Clin. Lower percentages of correction were seen when targeting the c.530 mutation for HDR correction in compound heterozygote 3, potentially due altered cell-state associated with the patients disease or the patients immunosuppressive drug regimen (Supplementary Table4). This is a preview of subscription content, access via your institution. Extended Data Fig. Bioz Stars score: 86/100, based on 1 PubMed citations. Open Access Murnane, J. P., Yezzi, M. J. 7 HDR-mediated correction of, Extended Data Fig. Sign up for the Nature Briefing newsletter what matters in science, free to your inbox daily. (b). Efficiency refers to the percentage of live cells in a culture expressing the knocked-in exogenous sequence (such as GFP). 4c). a, Except where noted otherwise, viability refers to the number of live cells in an experimental condition (expressed as a percentage) relative to an equivalent population that went through all protocol steps except for the actual electroporation (no electroporation control). Sci. CAS 5 Non-viral genome targeting using long ssDNA HDR templates and a Cas9 nickase. and JavaScript. h, Long ssDNA templates (~1.3kb) could be successfully combined with Cas9 nickases (D10A) for targeted integration, similar to linear dsDNA templates. Only the sorted NY-ESO-1+ TCR+ population demonstrated target cell killing (4:1 T cell to cancer cell ratio). n=2 (a, b, h, i) or n=8 (e, f) independent healthy donors. NSG mice (812 weeks old) were seeded with 1106 A375 cells (human melanoma cell line; NY-ESO-1 antigen+ and HLA-A*0201+) subcutaneously in a shaved flank. q, Target cell killing by non-viral TCR replacement T cells was due specifically to the NY-ESO-1-recognizing TCR+ cell population observed by flow cytometry after non-viral TCR replacement (Fig. By 144h, T cell to cancer cell ratios of less than 1:16 showed evidence of robust target cell killing. & Young, B. R. Recombination events during integration of transfected DNA into normal human cells. Below, we describe a protocol for CRISPR-Cas9 genome editing of human intestinal organoids cultured in IntestiCult Organoid Growth Medium (Human) (Catalog #06010) using the ArciTect CRISPR-Cas9 ribonucleoprotein (RNP)-based system and STEMCELL's Guide RNA Design Tool. DNA vectors are also the best option if you are looking to achieve stable transfection via incorporating your construct into the target cells genome. It is a highly efficient, non-viral method of transfection that is ideally suited for working with all cell types (although it is especially adept when using hard-to-transfect cell lines and primary cells). Methylation of the TSDR (Treg-cell-specific demethylated region) of FOXP3 intron 1 was analysed in the indicated sorted cell populations by bisulfite sequencing (Epigendx). J.H.E. USA 112, 1043710442 (2015). After annealing of an RT primer and reverse transcription, an RNADNA hybrid can form, which then can be transformed into a long ssDNA template by incubation in sodium hydroxide, which selectively degrades the RNA strand. Well, the ideal approach should offer high transfection efficiency, low toxicity and yield reproducible results. in n=4 (fi) independent healthy donors. 19. The exact molecular mixture has varied over time, as lipofection methods have improved. cost, time, facilities/expertise available etc. 1d). Graphs display mean (b, c, g, h) and/or individual donor values (bh) in n=2 independent healthy donors (bh). BMC Genomics g, Among the RNP and HDR template concentrations tested here, optimal GFP insertion into RAB11A was achieved at intermediate concentrations of the RNP and dsDNA HDRT. T cells from two donors were each electroporated twice with an 8 h rest in between electroporations. Protein transfection is used for specific applications, such as the introduction of Cas9 enzymes for gene editing via CRISPR, and the use of transcription factors to reprogram cells to generate iPSCs. ), grants from the Keck Foundation (A.M.), National Multiple Sclerosis Society (A.M.; CA 1074-A-21), gifts from J. Aronov, G. Hoskin, the Jeffrey Modell Foundation (A.M), and awards from the Burroughs Wellcome Fund (A.M.) and the Ressler Family Fund (C.P.S., J.S., A.R.). This can include using DNA, proteins, mRNA or non-coding RNAs, all of which will degrade over time or will be diluted out through cell division. Nature Viability was measured 2days after electroporation and GFP expression was measured at day4. For d, e and h, one representative donor is shown. Most commercial electroporation systems designed for transfection of primary mammalian cells (e.g., Lonza Nucleofector 4D, ThermoFisher Neon) use cuvettes for bulk, batch processing. 6i). ). CAS Efficient homology-directed gene editing by CRISPR/Cas9 in - Nature Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. If youd like to learn more about the different viruses used, download our free whitepaper: An Introduction to Transfection Methods Technical Reference Guide. g, TCR mispair analysis after retroviral delivery or non-viral TCR replacement of an NY-ESO-1-specific TCR in gated CD4+ or CD8+ T cells. e, Diagram of the genomic locus containing the first exon of RAB11A. This helps to ensure optimum transfection efficiency and cell viability. d, We determined whether the order of adding reagents influenced targeting efficiency and viability. XS Pichia and E.coli Expression Systems. h, Multiplex editing of combinatorial sets of genomic sites would support expanded research and therapeutic applications. Together, these studies provide preclinical evidence that non-viral genome targeting can enable rapid and flexible experimental manipulation and therapeutic engineering of primary human immune cells. This makes Nucleofection a powerful tool for many areas of research (see the figure on the right). More details on both transient and stable transfection are provided below. One HDR template, a C-terminal GFP fusion tag into the nuclear factor FBL, had consistently higher off-target expression across gRNAs, potentially due to a gene-trap effect as the 3 homology arm for FBL contains a splice-site acceptor followed by the final exon of FBL leading into the GFP fusion. This file contains Supplementary Notes 1-4, Raw data from electroporation pulse code optimizations (Extended Data Fig 2C), A list of HDR template, DNA primer, and gRNA sequences used in study, Mutation status and clinical phenotypes of members of a family with two distinct IL2RA coding mutations, including three patients with compound heterozygote mutations, Roth, T.L., Puig-Saus, C., Yu, R. et al. e, Mutation correction was possible in sorted Treg-like cells from the affected patients. Hornung, V. & Latz, E. Intracellular DNA recognition. 1 Development of non-viral genome targeting in primary human T cells. dsDNA HDR templates can be made easily by PCR followed by a SPRI purification to achieve a highly concentrated and pure product suitable for electroporation. Primary human T cells with two modifications were enriched by gating on the cells that had at least one modification, and this effect was consistent across multiple combinations of genomic loci. For each electroporation, 400 ng of the plasmid vector pMaxGFP (Lonza) in a volume of 2 l was placed in one well of a 96-well . ORDER YOUR CUSTOM HDR TEMPLATE Guide RNAs Cas9 nucleases Enhancers & controls CRISPR-Cas9 gRNA Guide RNAs (gRNAs) contain the target-specific sequence for guiding Cas9 protein to a genomic location. PubMed g, Quantification of different types of functional off-target integrations. Another practical consideration is that proteins must either be made and purified or purchased from a supplier both options can be costly, especially when compared to generating DNA or RNA substrates. High-voltage pulses of electricity are then applied to the mixture, which creates a potential difference across the cell membrane. Skene, P. J. A.M.F. Individual points represent replicates where the combination of the genes encoding the fluorescent proteins was varied (either GFP plus mCherry, GFP plus BFP, or mCherry plus BFP) as was the amount of the HDR template (36 g). In wells in which the RNP and the DNA HDR template were mixed together before adding the cells (1. Article The first is the NucleofectorTM Device, which offers a unique portfolio of electrical parameters, displayed as distinct programs that are adapted to the requirements of specific cell types. Article Google Scholar. Plasmid DNA is commonly used for transfection (although linear DNA can also be used). Preprint at https://doi.org/10.1101/178905 (2017). e, We looked for unintended non-homologous integrations with the non-viral system using an N-terminal GFP-RAB11A fusion construct that contained the endogenous RAB11A promoter sequence within its 5 homology arm. Mean and s.d. a, Unlike the gRNA targeting the c.800delA mutation at the C terminus of IL-2R (Extended Data Fig. The RNA used for transfection can take several forms, depending on the application. Protocol for Electroporation of Cas12a Ribonucleoprotein (RNP - NEB Present address: Chan Zuckerberg Biohub, San Francisco, CA, USA, Medical Scientist Training Program, University of California, San Francisco, San Francisco, CA, USA, Biomedical Sciences Graduate Program, University of California, San Francisco, San Francisco, CA, USA, Department of Microbiology and Immunology, University of California, San Francisco, San Francisco, CA, USA, Theodore L. Roth,Ruby Yu,Eric Shifrut,P. Jonathan Li,Joseph Hiatt,Victoria Tobin,David N. Nguyen,Kathrin Schumann&Alexander Marson, Diabetes Center, University of California, San Francisco, San Francisco, CA, USA, Theodore L. Roth,Ruby Yu,Eric Shifrut,P. Jonathan Li,Joseph Hiatt,Victoria Tobin,David N. Nguyen,Michael R. Lee,Amy L. Putnam,Kathrin Schumann&Alexander Marson, Innovative Genomics Institute, University of California, Berkeley, Berkeley, CA, USA, Department of Medicine, University of California at Los Angeles, Los Angeles, CA, USA, Cristina Puig-Saus,Justin Saco,Paige Krystofinski&Antoni Ribas, UCSF Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, San Francisco, CA, USA, Julia Carnevale,Alan Ashworth&Alexander Marson, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, CA, USA, Han Li,Jonathan S. Weissman&Manuel D. Leonetti, Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, CA, USA, HIV Dynamics and Replication Program, Vector Design and Replication Section, National Cancer Institute, Frederick, MD, USA, Department of Immunobiology, Yale School of Medicine, New Haven, CT, USA, Jeff W. Chen,Jean-Nicolas Schickel,Stephen H. Hughes&Eric Meffre, Division of Stem Cell Transplantation and Regenerative Medicine, Department of Pediatrics, Stanford University, Stanford, CA, USA, Laurence Pellerin,Rosa Bacchetta&Maria Grazia Roncarolo, Institute for Stem Cell Biology and Regenerative Medicine, Stanford University, Stanford, CA, USA, Section of Adult and Pediatric Endocrinology, Diabetes, and Metabolism, Departments of Medicine and Pediatrics, The University of Chicago, Chicago, IL, USA, Department of Human Genetics, The University of Chicago, Chicago, IL, USA, Gorka Alkorta-Aranburu&Daniela del Gaudio, Takara Bio USA, Inc, Mountain View, CA, USA, Hiroyuki Matsumoto,Montse Morell,Ying Mao,Baz Smith&Michael Haugwitz, Chan Zuckerberg Biohub, San Francisco, CA, USA, Min Cho,Andrew P. May&Alexander Marson, Mouse Genome Engineering Core Facility, Vice Chancellor for Research Office, University of Nebraska Medical Center, Omaha, NE, USA, Rolen M. Quadros&Channabasavaiah B. Gurumurthy, Department of Laboratory Medicine, University of California, San Francisco, San Francisco, CA, USA, Department of Pediatrics, Pathology, Yale School of Medicine, New Haven, CT, USA, Division of Immunology and Allergy, The Childrens Hospital of Philadelphia, Philadelphia, PA, USA, Department of Pediatrics, The Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA, Departments of Immunobiology and Internal Medicine, Yale University, New Haven, CT, USA, Department of Surgery, University of California, Los Angeles, Los Angeles, CA, USA, Department of Medical and Molecular Pharmacology, University of California, Los Angeles, Los Angeles, CA, USA, Jonsson Comprehensive Cancer Center, Los Angeles, CA, USA, Department of Medicine, University of California, San Francisco, San Francisco, CA, USA, You can also search for this author in
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