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  • ‹ Back to CRISPR/Cas9 delivery methods
  • Electroporation-grade Cas9 for editing in diverse cell types
  • CRISPR/Cas9 gene editing with AAV
  • CRISPR/Cas9 gesicles overview
  • Cas9 Gesicles—reduced off-target effects
  • sgRNA-Cas9 delivery to many cell types
  • Tet-inducible Cas9 for gene editing
Cas9-sgRNA gesicle production Cas9-sgRNA gesicle production
Home › Learning centers › Gene function › Gene editing › CRISPR/Cas9 delivery methods › CRISPR/Cas9 gesicles overview

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Cas9-sgRNA gesicle production Cas9-sgRNA gesicle production
Technology Overview

Gesicles enable CRISPR/Cas9-mediated gene editing with high efficiency and no additional footprint

The Guide-it CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesicles that will efficiently target your gene of interest

  • Gesicle production mechanism
  • Geiscle production system components
  • Gesicle production workflow
  • Ordering information
Videos Overview Components Workflow Conclusions

Videos  

Gesicle technology overview

Learn how gesicles are made and used, and the problems they were designed to overcome.

Gesicles gene editing webinar

Watch Dr. Baz Smith discuss gesicle technology and how it's used to perform CRISPR/Cas9-mediated gene editing.

Overview  

While CRISPR/Cas9 is a powerful technique for genome manipulation, two significant challenges remain: obtaining efficient delivery of the Cas9-sgRNA complex to all cell types, and leaving no additional footprint (i.e., persistent and elevated expression of Cas9 in target cells) that could lead to off-target effects. To address these challenges, we have developed a system of cell-derived nanovesicles called gesicles. Gesicles contain active Cas9 protein complexed with an sgRNA specific to a gene of interest. Thus, there is no persistent expression of Cas9, because no coding gene is present. Additionally, they are engineered with glycoproteins on their surface that mediate binding and fusion with the membrane of a wide range of target cells. These features enable gesicles to knock out genes with high efficiency and in a broader range of cell types than plasmid-based delivery methods. Finally, use of this method allows for control of the dose and duration of the Cas9-sgRNA complex in the cell, further reducing the chance of off-target effects.

Mechanism for producing gesicles to deliver a Cas9-sgRNA ribonucleoprotein complex

Gesicles are made by transfecting a 293T-based producer cell line with a vector containing an sgRNA for your target locus and our gesicle packaging mix, which contains everything you need in an optimized format. Loaded gesicles can then be collected from the supernatant and added to target cells for efficient editing with no footprint.

  1. 293T producer cells that have been transfected with our gesicle packaging mix and an sgRNA expression plasmid begin to form gesicles, stimulated by a nanovesicle-inducing glycoprotein
  2. The producer cells express active Cas9 endonuclease which forms a ribonucleoprotein (RNP) complex with your expressed sgRNA. Gesicle loading utilizes the iDimerize system, which enables inducible protein-protein interactions: The membrane-bound CherryPicker red fluorescent protein on the surface of the gesicle is tagged with one dimerization domain, and the Cas9 endonuclease contains another dimerization domain. A small heterodimerizer ligand is added to link these two domains together, thereby enriching the Cas9-sgRNA RNP complex into the gesicle.
  3. The red fluorescent protein-labeled gesicles are now loaded with active Cas9 complexed with your target sgRNA. Loaded gesicles pinch off from the producer cells and saturate the surrounding medium. Gesicles can be collected from the supernatant, yielding a concentrated stock of your Cas9-sgRNA gesicles. The gesicles can be used immediately on your target cells, or stored for over one year at –70°C.
  4. Harvested gesicles can be applied to a broad range of target cell types. In the presence of protamine sulfate, gesicles fuse to the membrane of the target cell via cell-surface glycoproteins and release the Cas9-sgRNA RNP complex into the cell. The membranes of cells that have been successfully targeted with gesicles are transiently labeled with red fluorescent protein. Critically, the absence of dimerizer ligand in the target-cell culture medium and the presence of a nuclear localization signal on the Cas9 endonuclease ensures that the complex dissociates from the red fluorescent protein and is transported to the nucleus. Once there, efficient targeted gene editing takes place. After editing occurs, the Cas9-sgRNA RNP complex is degraded over time by endogenous cell processes, leaving no remaining footprint that would be capable of producing off-target effects.

Components  

Gesicle production system components

The Guide-it CRISPR/Cas9 Gesicle Production System contains everything you need to easily produce gesicles that efficiently target your gene of interest. The kit includes lyophilized Xfect Transfection Reagent and gesicle packaging mixes which contain coding sequences for the nanovesicle-inducing glycoprotein, Cas9 endonuclease, and CherryPicker red fluorescent protein. A pre-linearized vector for cloning your target sgRNA and A/C Heterodimerizer for loading gesicles are also provided. Gesicle production can be performed in any 293T-based cell line, but we recommend the Gesicle Producer 293T Cell Line for optimal production results. Older or non-validated cell lines may produce gesicles less efficiently, thereby decreasing the gesicle concentration and subsequent editing efficiency.

Workflow  

Gesicle production system workflow

The Guide-it CRISPR/Cas9 Gesicle Production System workflow is simple and straightforward. Your sgRNA for a gene of interest is cloned into the provided linearized expression plasmid, pGuide-it-sgRNA1. Next, this cloned plasmid is mixed with dH2O and the Guide-It CRISPR/Cas9 Gesicle Packaging Mixes 1 and 2. Following a 10-minute incubation, the complete nanoparticle mix is applied to 293T-based producer cells in the presence of the provided A/C Heterodimerizer ligand. After 48–72 hours, gesicles containing active Cas9 protein complexed with your sgRNA can be collected and concentrated via centrifugation. Gesicles can be used immediately, or stored at –70°C for more than one year.

Conclusions  

The Guide-it CRISPR/Cas9 Gesicle Production System is a complete kit that enables highly efficient gene editing without any additional footprint. This kit contains all of the reagents necessary to prepare custom gesicles to deliver Cas9 and a user-defined, gene-specific guide sequence. Gesicle-based delivery of Cas9 protein and sgRNA using this system results in successful genome editing in a broader range of cell types compared to delivery by plasmid transfection. Critically, gesicles do not result in persistence of the Cas9 endonuclease, which in turn reduces the chances of off-target effects and provides precise control of the dose and timing of gene editing.

Related products

Cat. # Product Size License Quantity Details
632612 pGuide-it-sgRNA1 Vector System 1 System USD $325.00

License Statement

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272 This product (“Product”) and its use, is the subject of U.S. Patents 8,697,359 and 8,771,945 and pending U.S. Patent applications. The purchase of the Product conveys to the buyer the non-transferable right to use Product(s) purchased from Takara Bio USA, Inc. or its Affiliates, and any progeny, modification or derivative of a Product, or any cell or animal made or modified through use of a Product, or any progeny, modification or derivative of such cell or animal (“Related Material”), solely for research conducted by the buyer in accordance with all of the following requirements. No right is given to use this Product or Related Material for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. The buyer shall not sell or otherwise transfer Products (including without limitation any material that contains a Licensed Product in whole or part) or any Related Material to any other person or entity, or use Products or any Related Material to perform services for the benefit of any other person or entity, (ii) the buyer shall use only the purchased amount of the Products and components of the Products, and shall use any Related Material, only for its internal research and not for (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis (“Commercial Purpose”), (iii) the buyer shall use Licensed Products and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations, and (iv) the buyer shall indemnify, defend and hold harmless MIT, Harvard and The Broad and their current and former trustees, directors, officers, faculty, affiliated investigators, students, employees, and agents and their respective successors, heirs and assigns (“Indemnitees”), against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the buyer, or any breach of the rights granted hereunder by the buyer.
391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.
396 Sigma-Aldrich CRISPR Use License Agreement This Product and its use are the subject of one or more of the following issued patents and patent applications: Australia Patent Nos. 2013355214; 2017204031; and 2018229489; Canada Patent Nos. 2,891,347 and 2,977,152; China Patent No. CN105142669; European Patent Nos. EP 2 928 496 B1; EP 3 138 910 B1, 3 138 911 B1, EP 3 138 912 B1, EP 3 360 964 B1, EP 3 363 902 B1; Israel Patent No. IL238856; Singapore Patent No. 11201503824S; South Korea Patent Nos. 10-1844123 and 10-2006880; and U.S. Patent Application Serial Nos. 15/188,911; 15/188,924; 15/188,927; 15/188,931; and 15/456,204 (the “Patent Rights”). The purchase of this Product conveys to you (the “Buyer”) the NON-TRANSFERABLE right to use the Product for Licensed Research Use (see definition below) subject to the conditions set out in this License Agreement. 1. “Licensed Research Use” means any use for research purposes, except: (i) Buyer may not sell or otherwise transfer the Product (including without limitation any material that contains the Product in whole or part) or any Related Material to any other third party (except that you may transfer the Product, or any Related Material to a bona fide collaborator or contract research organization), or use the Products or any Related Material to perform services for the benefit of any other third party; (ii) Buyer may use only the purchased amount of the Product and components of the Product, and shall use any Related Material, only for your internal research within the Field, and not for any Commercial Purposes; (iii) Buyer shall use the Product and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations; and (iv) the Buyer shall indemnify, defend, and hold harmless SIGMA and their current and former directors, officers, employees and agents, and their respective successors, heirs and assigns (the “Indemnities”) against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the Buyer hereunder or any breach of this License Agreement by such Buyer. 2. For purposes of Section 1 above, the following definitions shall apply: “Commercial Purposes” means (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis. “Field” means use as a research tool for research purposes; provided, however, that notwithstanding the foregoing, the Field shall expressly exclude (a) any in vivo and ex vivo human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) the creation of transgenic rodent models and/or derivatives thereof (including, but not limited to, rodents’ cells and rodents’ organs) by for-profit entities, (c) any in vivo veterinary or livestock use, or non-research agricultural use, or (d) use as a testing service, therapeutic or diagnostic for humans or animals. “Related Materials” means any progeny, modification or derivative of a Product. 3. Your right to use the Product will terminate immediately if you fail to comply with these terms and conditions. You shall, upon such termination of your rights, destroy all Product, Related Materials, and components thereof in your control, and notify SIGMA of such in writing. For information on purchasing a license to this Product for purposes other than Licensed Research Use, contact your local SIGMA sales representative, or call +1 800-325-3010.

The pGuide-it-sgRNA1 Vector System is a complete system for cloning and expression of a target-specific single guide RNA (sgRNA) for mammalian gene modification studies using CRISPR/Cas9 technology. The pGuide-it-sgRNA1 Vector is used to express a target-specific sgRNA. This vector is pre-linearized for simple insertion of a target sequence for sgRNA expression from the human U6 promoter. It does not contain a selectable marker or fluorescence reporter. pGuide-it-sgRNA1 Vector can be used for transient transfection of mammalian cells. This kit also contains all of the components needed for cloning, including ligation mix, necessary controls, and Stellar Competent Cells.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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632612: pGuide-it-sgRNA1 Vector System

632612: pGuide-it-sgRNA1 Vector System
632613 Guide-it™ CRISPR/Cas9 Gesicle Production System 1 System USD $713.00

License Statement

ID Number  
44 The DsRed-Monomer and the Fruit Fluorescent Proteins are covered by one or more of the following U.S. Patents: 7,671,185, 7,9110,714 and 8,664,471.
69 The DsRed-Monomer, DsRed Express, E2-Crimson and the Fruit Fluorescent Proteins are covered by one or more of the following U.S. Patents: 7,250,298; 7,671,185; 7,910,714; 8,664,471 and 8,679,749.
72 Living Colors Fluorescent Protein Products:

Not-For-Profit Entities: Orders may be placed in the normal manner by contacting your local representative or Takara Bio USA, Inc. Customer Service. Any and all uses of this product will be subject to the terms and conditions of the Non-Commercial Use License Agreement (the “Non-Commercial License”), a copy of which can be found below. As a condition of sale of this product to you, and prior to using this product, you must agree to the terms and conditions of the Non-Commercial License. Under the Non-Commercial License, Takara Bio USA, Inc. grants Not-For-Profit Entities a non-exclusive, non-transferable, non-sublicensable and limited license to use this product for internal, non-commercial scientific research use only. Such license specifically excludes the right to sell or otherwise transfer this product, its components or derivatives thereof to third parties. No modifications to the product may be made without express written permission from Takara Bio USA, Inc. Any other use of this product requires a different license from Takara Bio USA, Inc. For license information, please contact a licensing representative by phone at 650.919.7320 or by e-mail at licensing@takarabio.com.

For-Profit Entities wishing to use this product are required to obtain a license from Takara Bio USA, Inc. For license information, please contact a licensing representative by e-mail at licensing@takarabio.com.

Not-For-Profit Non-Commercial Use License:
A copy of the Guide-it™ CRISPR/Cas9 Gesicle Production System product License Agreement can be found by clicking here.
272 This product (“Product”) and its use, is the subject of U.S. Patents 8,697,359 and 8,771,945 and pending U.S. Patent applications. The purchase of the Product conveys to the buyer the non-transferable right to use Product(s) purchased from Takara Bio USA, Inc. or its Affiliates, and any progeny, modification or derivative of a Product, or any cell or animal made or modified through use of a Product, or any progeny, modification or derivative of such cell or animal (“Related Material”), solely for research conducted by the buyer in accordance with all of the following requirements. No right is given to use this Product or Related Material for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. The buyer shall not sell or otherwise transfer Products (including without limitation any material that contains a Licensed Product in whole or part) or any Related Material to any other person or entity, or use Products or any Related Material to perform services for the benefit of any other person or entity, (ii) the buyer shall use only the purchased amount of the Products and components of the Products, and shall use any Related Material, only for its internal research and not for (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis (“Commercial Purpose”), (iii) the buyer shall use Licensed Products and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations, and (iv) the buyer shall indemnify, defend and hold harmless MIT, Harvard and The Broad and their current and former trustees, directors, officers, faculty, affiliated investigators, students, employees, and agents and their respective successors, heirs and assigns (“Indemnitees”), against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the buyer, or any breach of the rights granted hereunder by the buyer.
225 Gesicle Technology. This product and its use are the subject of European patents and patent applications related to EP09306091, owned by Inserm Transfert. For research use only – not for therapeutic or diagnostic use in humans. No rights granted other that the right to use for research purposes.

In addition, Inserm Transfert is the owner of European patent application EP09306092.9 relating to the use of microvesicles for cell engineering purposes. The products should not be used to develop, make, have made, use and sell or otherwise distribute any product, composition, method, service or process for commercial services that would infringe said patent application, unless a license is negotiated with Inserm Transfert, at 7 rue Watt, 75013 Paris, France (www.inserm-transfert.fr).
391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.
396 Sigma-Aldrich CRISPR Use License Agreement This Product and its use are the subject of one or more of the following issued patents and patent applications: Australia Patent Nos. 2013355214; 2017204031; and 2018229489; Canada Patent Nos. 2,891,347 and 2,977,152; China Patent No. CN105142669; European Patent Nos. EP 2 928 496 B1; EP 3 138 910 B1, 3 138 911 B1, EP 3 138 912 B1, EP 3 360 964 B1, EP 3 363 902 B1; Israel Patent No. IL238856; Singapore Patent No. 11201503824S; South Korea Patent Nos. 10-1844123 and 10-2006880; and U.S. Patent Application Serial Nos. 15/188,911; 15/188,924; 15/188,927; 15/188,931; and 15/456,204 (the “Patent Rights”). The purchase of this Product conveys to you (the “Buyer”) the NON-TRANSFERABLE right to use the Product for Licensed Research Use (see definition below) subject to the conditions set out in this License Agreement. 1. “Licensed Research Use” means any use for research purposes, except: (i) Buyer may not sell or otherwise transfer the Product (including without limitation any material that contains the Product in whole or part) or any Related Material to any other third party (except that you may transfer the Product, or any Related Material to a bona fide collaborator or contract research organization), or use the Products or any Related Material to perform services for the benefit of any other third party; (ii) Buyer may use only the purchased amount of the Product and components of the Product, and shall use any Related Material, only for your internal research within the Field, and not for any Commercial Purposes; (iii) Buyer shall use the Product and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations; and (iv) the Buyer shall indemnify, defend, and hold harmless SIGMA and their current and former directors, officers, employees and agents, and their respective successors, heirs and assigns (the “Indemnities”) against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the Buyer hereunder or any breach of this License Agreement by such Buyer. 2. For purposes of Section 1 above, the following definitions shall apply: “Commercial Purposes” means (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis. “Field” means use as a research tool for research purposes; provided, however, that notwithstanding the foregoing, the Field shall expressly exclude (a) any in vivo and ex vivo human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) the creation of transgenic rodent models and/or derivatives thereof (including, but not limited to, rodents’ cells and rodents’ organs) by for-profit entities, (c) any in vivo veterinary or livestock use, or non-research agricultural use, or (d) use as a testing service, therapeutic or diagnostic for humans or animals. “Related Materials” means any progeny, modification or derivative of a Product. 3. Your right to use the Product will terminate immediately if you fail to comply with these terms and conditions. You shall, upon such termination of your rights, destroy all Product, Related Materials, and components thereof in your control, and notify SIGMA of such in writing. For information on purchasing a license to this Product for purposes other than Licensed Research Use, contact your local SIGMA sales representative, or call +1 800-325-3010.
405 This product is protected by U.S. Patent Nos. 9593356 and 10793828 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
259 This product is protected by Japanese Patent No. 6454352 and corresponding U.S. pending patent and other foreign patents pending. For further license information, please contact a Takara Bio USA licensing representative by email at  licensing@takarabio.com.

The Guide-it CRISPR/Cas9 Gesicle Production System is a system for producing high yields of target-specific CRISPR/Cas9 gesicles for gene editing. Gesicles are cell-derived nanovesicles used to deliver macromolecular cargoes to a broad range of target cells, including cells that are difficult to transfect with plasmids. The nanovesicles are produced in a Gesicle Producer 293T Cell Line (Cat. No. 632617) via co-overexpression of packaging mix components, which include a nanovesicle-inducing glycoprotein and a protein that is displayed on the cell surface that mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of additional macromolecular cargoes, in this case the Cas9 protein from Streptococcus pyogenes and a target-specific guide RNA (sgRNA), results in incorporation of the Cas9/sgRNA complex within the gesicles. After the resulting Cas9/sgRNA gesicles are harvested and applied to your target cells in the presence of protamine sulfate, they will efficiently enter the cells and mediate gene editing. This system provides the components needed to clone and express your target-specific guide RNA, and packaging reagents to produce CRISPR/Cas9 gesicles. It is essential to use the pGuide-it-sgRNA1 Vector for expression and packaging of your guide RNA, since using other commonly used guide RNA vectors will not result in effective Cas9/sgRNA complexes.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

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The use of gesicles decreases off-target effects

The use of gesicles decreases off-target effects

The use of gesicles decreases off-target effects. HEK 293T cells were either simultaneously cotransfected with plasmids encoding Cas9 DNA and a sgRNA against EMX1, or treated with gesicles loaded with Cas9-sgRNA ribonucleoprotein complexes. After 72 hr, the EMX1 gene and a potential off-target locus (off-target 4) were analyzed using the Guide-it Mutation Detection Kit (Cat. # 631443). With the gesicles, no off-target effect could be detected (top panel). Sequencing data for the different clones were aligned with the underlined wild-type sequence, revealing a range of deletions and insertions (indels; highlighted in red). Gesicles correctly edited the EMX1 gene with no off-target effects, whereas plasmid cotransfection resulted in indels in both the target site, EMX1, as well as off-target site 4 (bottom panel).

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Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles

Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles
Knockout efficiency of fluorescent reporter by Cas9-sgRNA protein complexes delivered to various cell types using gesicles. Cell lines were created that contained an integrated ZsGreen1 fluorescent protein expression cassette. In this system, successful Cas9-mediated cleavage can be measured by loss of ZsGreen1 expression. These cell lines were treated with gesicles loaded with Cas9-sgRNA protein complexes (with the sgRNA generated against ZsGreen1), and then analyzed by flow cytometry. Cas9-sgRNA protein complex delivery and ZsGreen1 knockout via gesicles was efficient and comparable to plasmid-based delivery in easier-to-transfect cell types (left graph) and surpassed the results achieved via plasmid-based delivery in harder-to-transfect cell types (right graph).

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Gesicle production overview for delivery of Cas9-sgRNA

Gesicle production overview for delivery of Cas9-sgRNA
Gesicle production overview for delivery of Cas9-sgRNA. (Step 1) Gesicle formation is induced by glycoproteins on the surface of 293T producer cells that have been cotransfected with our gesicle packaging mix and a target-specific guide RNA plasmid. (Step 2) Utilizing the iDimerize system, a small ligand is added to load the Cas9-sgRNA ribonucleoprotein complex into the gesicle through interaction with the membrane-bound CherryPicker red fluorescent protein on the gesicle surface. (Step 3) Loaded and red fluorescent protein-labeled gesicles pinch off from the producer cells and are collected from the supernatant, yielding a concentrated stock of Cas9-sgRNA gesicles. (Step 4) Harvested gesicles can be applied to a broad range of target cell types, to which they fuse, transiently labeling the cells red and releasing the Cas9-sgRNA complex into the cell. The presence of an NLS on the Cas9 protein and the absence of the dimerizer ligand in your cell culture medium ensures that the complex is transported to the nucleus after dissociating from the red fluorescent protein.

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The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

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Editing efficiency is increased by an improved sgRNA scaffold design in gesicles

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles

Editing efficiency is increased by an improved sgRNA scaffold design in gesicles. HT1080 cells containing an integrated fluorescent protein expression cassette were transfected with a plasmid encoding for Cas9 and AcGFP1-specific sgRNA or treated with gesicles. Both delivery methods were tested using either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. The optimized sgRNA scaffold had no effect on editing efficiency for plasmid-based delivery. However, the optimized sgRNA scaffold increased knockout efficiency by 36.4% for gesicles. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

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Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect

Using an improved sgRNA scaffold design in gesicles enables a dose-dependent increase in knockout effect. HT1080 cells containing an integrated fluorescent protein expression cassette were treated with 5 µl, 10 µl, 20 µl, or 30 µl of gesicles produced with either the traditional or an optimized, sgRNA scaffold targeting AcGFP1. The optimized scaffold has an extension of the Cas9-binding hairpin and removes four consecutive uracils. The knockout efficiency was measured six days later by flow cytometry analysis. Editing efficiency using the traditional scaffold was low, regardless of gesicle dose. However, gesicles using the optimized scaffold demonstrated a dose-dependent increase in knockout up to 59%. Thus, only the pGuide-it-sgRNA1 vector containing the optimized scaffold is recommended for gesicle production.

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Optimal sgRNA design strategies when using gesicles

Optimal sgRNA design strategies when using gesicles
Optimal sgRNA design strategies when using gesicles. While evaluating potential sgRNAs for knockout of different genes, several optimal strategies were identified. First, having a guanine in the first position of the target sequence provided the highest knockout efficiency. Additionally, having either an adenine or thymine in the seventeenth position of the target sequence was beneficial. Utilizing these two design principles when producing gesicles can improve overall editing efficiency in target cells.

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Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes

Efficient knockout of an endogenous protein (CD81) using gesicles containing Cas9-sgRNA complexes. The cell-surface protein receptor CD81 was knocked out in Jurkat cells using either plasmid cotransfection of Cas9 DNA and sgRNA or gesicles preloaded with a Cas9-sgRNA ribonucleoprotein complex. The knockout efficiency was measured six days later via antibody labeling of the membrane receptor followed by flow cytometry analysis. Results for delivery via gesicles were significantly greater than results achieved with plasmid transfection.

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6501_gesicle_tube

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Gesicles utilize an optimized sgRNA scaffold

Gesicles utilize an optimized sgRNA scaffold
Gesicles utilize an optimized sgRNA scaffold. The traditional sgRNA scaffold was modified by extending the Cas9-binding hairpin and removing four consecutive uracils. This optimized scaffold is critical for successful gesicle-based editing due to improved knockout efficiency.

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Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

632616 Guide-it™ CRISPR/Cas9 Gesicle Packaging Set 10 Rxns USD $610.00

License Statement

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272 This product (“Product”) and its use, is the subject of U.S. Patents 8,697,359 and 8,771,945 and pending U.S. Patent applications. The purchase of the Product conveys to the buyer the non-transferable right to use Product(s) purchased from Takara Bio USA, Inc. or its Affiliates, and any progeny, modification or derivative of a Product, or any cell or animal made or modified through use of a Product, or any progeny, modification or derivative of such cell or animal (“Related Material”), solely for research conducted by the buyer in accordance with all of the following requirements. No right is given to use this Product or Related Material for any other purpose, including, but not limited to, use in drugs, in vitro diagnostic purposes, therapeutics, or in humans. The buyer shall not sell or otherwise transfer Products (including without limitation any material that contains a Licensed Product in whole or part) or any Related Material to any other person or entity, or use Products or any Related Material to perform services for the benefit of any other person or entity, (ii) the buyer shall use only the purchased amount of the Products and components of the Products, and shall use any Related Material, only for its internal research and not for (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis (“Commercial Purpose”), (iii) the buyer shall use Licensed Products and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations, and (iv) the buyer shall indemnify, defend and hold harmless MIT, Harvard and The Broad and their current and former trustees, directors, officers, faculty, affiliated investigators, students, employees, and agents and their respective successors, heirs and assigns (“Indemnitees”), against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the buyer, or any breach of the rights granted hereunder by the buyer.
225 Gesicle Technology. This product and its use are the subject of European patents and patent applications related to EP09306091, owned by Inserm Transfert. For research use only – not for therapeutic or diagnostic use in humans. No rights granted other that the right to use for research purposes.

In addition, Inserm Transfert is the owner of European patent application EP09306092.9 relating to the use of microvesicles for cell engineering purposes. The products should not be used to develop, make, have made, use and sell or otherwise distribute any product, composition, method, service or process for commercial services that would infringe said patent application, unless a license is negotiated with Inserm Transfert, at 7 rue Watt, 75013 Paris, France (www.inserm-transfert.fr).
63 Use of this product is covered by one or more of the following U.S. Patent Nos. and corresponding patent claims outside the U.S.: 6,998,115; 7,427,394. This product is intended for research purposes only. It may not be used for (i) any human or veterinary use, including without limitation therapeutic and prophylactic use, (ii) any clinical use, including without limitation diagnostic use, (iii) screening of chemical and/or biological compounds for the identification of pharmaceutically active agents (including but not limited to screening of small molecules), target validation, preclinical testing services, or drug development. Any use of this product for any of the above mentioned purposes requires a license from the Massachusetts Institute of Technology.
69 The DsRed-Monomer, DsRed Express, E2-Crimson and the Fruit Fluorescent Proteins are covered by one or more of the following U.S. Patents: 7,250,298; 7,671,185; 7,910,714; 8,664,471 and 8,679,749.
44 The DsRed-Monomer and the Fruit Fluorescent Proteins are covered by one or more of the following U.S. Patents: 7,671,185, 7,9110,714 and 8,664,471.
72 Living Colors Fluorescent Protein Products:

Not-For-Profit Entities: Orders may be placed in the normal manner by contacting your local representative or Takara Bio USA, Inc. Customer Service. Any and all uses of this product will be subject to the terms and conditions of the Non-Commercial Use License Agreement (the “Non-Commercial License”), a copy of which can be found below. As a condition of sale of this product to you, and prior to using this product, you must agree to the terms and conditions of the Non-Commercial License. Under the Non-Commercial License, Takara Bio USA, Inc. grants Not-For-Profit Entities a non-exclusive, non-transferable, non-sublicensable and limited license to use this product for internal, non-commercial scientific research use only. Such license specifically excludes the right to sell or otherwise transfer this product, its components or derivatives thereof to third parties. No modifications to the product may be made without express written permission from Takara Bio USA, Inc. Any other use of this product requires a different license from Takara Bio USA, Inc. For license information, please contact a licensing representative by phone at 650.919.7320 or by e-mail at licensing@takarabio.com.

For-Profit Entities wishing to use this product are required to obtain a license from Takara Bio USA, Inc. For license information, please contact a licensing representative by e-mail at licensing@takarabio.com.

Not-For-Profit Non-Commercial Use License:
A copy of the Guide-it™ CRISPR/Cas9 Gesicle Packaging Set product License Agreement can be found by clicking here.
391 LIMITED USE LABEL LICENSE: RESEARCH USE ONLY Notice to Purchaser: This product is the subject to a license granted to Takara Bio USA, Inc. and its Affiliates from Caribou Biosciences, Inc., and this product is transferred to the end-user purchaser (“Purchaser”) subject to a “Limited Use Label License” conveying to the Purchaser a limited, non-transferable right to use the product, solely as provided to Purchaser, together with (i) progeny or derivatives of the product generated by the Purchaser (including but not limited to cells), and (ii) biological material extracted or derived from the product or its corresponding progeny or derivatives (including but not limited to cells) (collectively, the product, and (i) and (ii) are referred to as (“Material”) only to perform internal research for the sole benefit of the Purchaser. The Purchaser cannot sell or otherwise transfer Material to a third party or otherwise use the Material for any Excluded Use. “Excluded Use” means any and all: (a) commercial activity including, but not limited to, any use in manufacturing (including but not limited to cell line development for purposes of bioproduction), product testing, or quality control; (b) preclinical or clinical testing or other activity directed toward the submission of data to the U.S. Food and Drug Administration, or any other regulatory agency in any country or jurisdiction where the active agent in such studies comprises the Material; (c) use to provide a service, information, or data to a third party; (d) use for human or animal therapeutic, diagnostic, or prophylactic purposes or as a product for therapeutics, diagnostics, or prophylaxis; (e) activity in an agricultural field trial or any activity directed toward the submission of data to the U.S. Department of Agriculture or any other agriculture regulatory agency; (f) high throughput screening drug discovery purposes (i.e., the screening of more than 10,000 experiments per day) as well as scale-up production activities for commercialization; (g) modification of human germline, including editing of human embryo genomes (with the sole exception of editing human embryonic stem (ES) cell lines for research purposes) or reproductive cells; (h) self-editing; and/or (i) stimulation of biased inheritance of a particular gene or trait or set of genes or traits (“gene drive”). It is the Purchaser’s responsibility to use the Material in accordance with all applicable laws and regulations. For information on obtaining additional rights, including commercial rights, please contact licensing@cariboubio.com or Caribou Biosciences, Inc., 2929 7th Street, Suite 105, Berkeley, CA 94710 USA, Attn: Licensing.
396 Sigma-Aldrich CRISPR Use License Agreement This Product and its use are the subject of one or more of the following issued patents and patent applications: Australia Patent Nos. 2013355214; 2017204031; and 2018229489; Canada Patent Nos. 2,891,347 and 2,977,152; China Patent No. CN105142669; European Patent Nos. EP 2 928 496 B1; EP 3 138 910 B1, 3 138 911 B1, EP 3 138 912 B1, EP 3 360 964 B1, EP 3 363 902 B1; Israel Patent No. IL238856; Singapore Patent No. 11201503824S; South Korea Patent Nos. 10-1844123 and 10-2006880; and U.S. Patent Application Serial Nos. 15/188,911; 15/188,924; 15/188,927; 15/188,931; and 15/456,204 (the “Patent Rights”). The purchase of this Product conveys to you (the “Buyer”) the NON-TRANSFERABLE right to use the Product for Licensed Research Use (see definition below) subject to the conditions set out in this License Agreement. 1. “Licensed Research Use” means any use for research purposes, except: (i) Buyer may not sell or otherwise transfer the Product (including without limitation any material that contains the Product in whole or part) or any Related Material to any other third party (except that you may transfer the Product, or any Related Material to a bona fide collaborator or contract research organization), or use the Products or any Related Material to perform services for the benefit of any other third party; (ii) Buyer may use only the purchased amount of the Product and components of the Product, and shall use any Related Material, only for your internal research within the Field, and not for any Commercial Purposes; (iii) Buyer shall use the Product and any Related Material in compliance with all applicable laws and regulations, including without limitation applicable human health and animal welfare laws and regulations; and (iv) the Buyer shall indemnify, defend, and hold harmless SIGMA and their current and former directors, officers, employees and agents, and their respective successors, heirs and assigns (the “Indemnities”) against any liability, damage, loss, or expense (including without limitation reasonable attorneys’ fees and expenses) incurred by or imposed upon any of the Indemnitees in connection with any claims, suits, investigations, actions, demands or judgments arising out of or related to the exercise of any rights granted to the Buyer hereunder or any breach of this License Agreement by such Buyer. 2. For purposes of Section 1 above, the following definitions shall apply: “Commercial Purposes” means (a) the practice, performance or provision of any method, process or service, or (b) the manufacture, sale, use, distribution, disposition or importing of any product, in each case (a) or (b) for consideration, or on any other commercial basis. “Field” means use as a research tool for research purposes; provided, however, that notwithstanding the foregoing, the Field shall expressly exclude (a) any in vivo and ex vivo human or clinical use, including, without limitation, any administration into humans or any diagnostic or prognostic use, (b) the creation of transgenic rodent models and/or derivatives thereof (including, but not limited to, rodents’ cells and rodents’ organs) by for-profit entities, (c) any in vivo veterinary or livestock use, or non-research agricultural use, or (d) use as a testing service, therapeutic or diagnostic for humans or animals. “Related Materials” means any progeny, modification or derivative of a Product. 3. Your right to use the Product will terminate immediately if you fail to comply with these terms and conditions. You shall, upon such termination of your rights, destroy all Product, Related Materials, and components thereof in your control, and notify SIGMA of such in writing. For information on purchasing a license to this Product for purposes other than Licensed Research Use, contact your local SIGMA sales representative, or call +1 800-325-3010.
405 This product is protected by U.S. Patent Nos. 9593356 and 10793828 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
259 This product is protected by Japanese Patent No. 6454352 and corresponding U.S. pending patent and other foreign patents pending. For further license information, please contact a Takara Bio USA licensing representative by email at  licensing@takarabio.com.

Gesicles are cell-derived nanovesicles used to deliver macromolecular cargoes to a broad range of target cells, including cells that are difficult to transfect with plasmids. The nanovesicles generated using the Guide-it CRISPR/Cas9 Gesicle Packaging Set are produced in a Gesicle Producer 293T Cell Line (Cat. No. 632617) via co-overexpression of packaging mix components, which include a nanovesicle-inducing glycoprotein and a protein that is displayed on the cell surface and mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of additional macromolecular cargoes, in this case the Cas9 protein from Streptococcus pyogenes and a target-specific guide RNA (sgRNA), results in incorporation of the Cas9/sgRNA complex within the gesicles. After the resulting Cas9/sgRNA gesicles are harvested and applied to your target cells in the presence of protamine sulfate, they will efficiently enter the cells and mediate gene editing. The Guide-it CRISPR/Cas9 Gesicles Packaging Mixes 1 and 2 are each supplied prealiquoted and lyophilized in single vials containing Xfect Transfection Reagent and an optimized combination of gesicle-producing components. Gesicles are produced by sequentially reconstituting the lyophilized mixtures with your guide RNA vector of choice in sterile water and transfecting the sample into Gesicle Producer 293T cells according to the protocol in the Guide-it CRISPR/Cas9 Gesicle Production System User Manual. It is essential to use the pGuide-itsgRNA1 Vector for expression/packaging of your guide RNA, since using other commonly used guide RNA vectors will not result in effective Cas9/sgRNA complexes.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

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Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

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632616: Guide-it CRISPR/Cas9 Gesicle Packaging Set

632616: Guide-it CRISPR/Cas9 Gesicle Packaging Set
632617 Gesicle Producer 293T Cell Line 1 mL USD $387.00

License Statement

ID Number  
406 This product is the subject of a technology license agreement for internal research use only. Use of this product other than for research use may require additional licenses. Information on license restrictions or for uses other than research may be obtained by contacting licensing@takarabio.com.
330 This product is the subject of a technology license agreement for internal research use only. Use of this product other than for research use may require additional licenses. Information on license restrictions or for uses other than research may be obtained by contacting licensing@takarabio.com.
405 This product is protected by U.S. Patent Nos. 9593356 and 10793828 and corresponding foreign patents. Additional patents are pending. For further license information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The Gesicle Producer 293T Cell Line is a subclone of the transformed human embryonic kidney cell line, HEK 293, which is highly transfectable and supports high levels of protein expression. The cell line also constitutively expresses the simian virus 40 (SV40) large T antigen. Gesicles are produced in these cells via co-overexpression of packaging mix components, which include a nanovesicle-inducing glycoprotein and a protein that is displayed on the cell surface and mediates binding and fusion with the cellular membrane of target cells. Simultaneous overexpression of another protein cargo can result in incorporation of that protein within the gesicles. When combined with a gesicle production system, these cells are capable of producing high gesicle yields.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells

Cas9 protein delivery in culture cells. Immunohistochemistry was performed on RPE cells stably expressing ZsGreen1 and treated with Cas9 gesicles. Cells were stained 12 hr after addition of gesicles. Cas9 was detected using the Guide-it Cas9 Polyclonal Antibody (Cat. # 632607) together with the Alexa 350-conjugated anti-rabbit IgG secondary antibody. Red fluorescence from the CherryPicker fluorescent protein could also be detected in the cells

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Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells

Successful knockout of CD81 in hiPS cells. Gesicles containing Cas9-sgRNA complexes designed to target human CD81 were harvested and added to Cellartis Human iPS Cell Line 18 (hiPSC ChiPSC18), cultured in Cellartis DEF-CS Culture System for 6 and 24 hr, and then cultured in gesicle-free DEF-CS culture media for an additional 7 days. The surface expression of CD81 on gesicle-treated cells and untreated (control) cells was determined via flow cytometry analysis using FITC-labeled antibodies against CD81. Panel A. CD81 negative (left) and positive (right) labeling controls with hiPSC ChiPSC18 cells. Panel B. DEF-hiPSC ChiPSC18 cells after 6 hr (left) and 24 hr (right) of gesicle treatment, labeled with anti-CD81 (FITC) antibodies.

631318 Xfect™ Transfection Reagent 300 Rxns USD $561.00

License Statement

ID Number  
63 Use of this product is covered by one or more of the following U.S. Patent Nos. and corresponding patent claims outside the U.S.: 6,998,115; 7,427,394. This product is intended for research purposes only. It may not be used for (i) any human or veterinary use, including without limitation therapeutic and prophylactic use, (ii) any clinical use, including without limitation diagnostic use, (iii) screening of chemical and/or biological compounds for the identification of pharmaceutically active agents (including but not limited to screening of small molecules), target validation, preclinical testing services, or drug development. Any use of this product for any of the above mentioned purposes requires a license from the Massachusetts Institute of Technology.

Xfect is a transfection reagent that creates biodegradable nanoparticles that permit superior transfection efficiency of plasmid DNA into mammalian cells. Transfections can be carried out entirely in the presence of serum.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent

High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent
High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent.

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Transfection of Jurkat cells

Transfection of Jurkat cells

Transfection of Jurkat cells. Suspension cells such as Jurkat cells are notoriously very hard to transfect, but using the Xfect Transfection Reagent, you can achieve almost 30% efficiency. The transfection efficiency using the leading competitor product, on the other hand, is no higher than background.

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Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent

Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent

Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent. The Xfect Reagent and Product L were each used according to their respective protocols to transfect HeLa cells with increasing amounts of plasmid DNA encoding the Living Colors fluorescent protein, AcGFP1. 48 hr posttransfection, AcGFP1 expression was assessed by flow cytometry in order to determine transfection efficiency and cell viability was assessed by trypan dye exclusion assay.

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Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent

Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent
Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent.

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Share without contaminating

Share without contaminating

Share without contaminating. Xfect Transfection Reagent is packaged in convenient, separable kits, so if you purchase the 100 reaction kit (2 x 50 rxns) or 300 reaction kit (3 x 100 rxns) you can share with your less-than-careful colleagues without fear.

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The simple Xfect Transfection Reagent protocol is completely serum-compatible

The simple Xfect Transfection Reagent protocol is completely serum-compatible
The simple Xfect Transfection Reagent protocol is completely serum-compatible.

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Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here

Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here
Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here.

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631318: Xfect Transfection Reagent

631318: Xfect Transfection Reagent
631443 Guide-it™ Mutation Detection Kit 100 Rxns USD $506.00

The Guide-it Mutation Detection Kit contains all the reagents needed for PCR-based identification of insertions or deletions generated during cellular non-homologous end joining (NHEJ) repair. The first step is the amplification of the putative target sequence directly from cells. This kit uses Terra PCR Direct Polymerase Mix and Buffer, so there is no need to extract genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets for cleavage by the Guide-it Resolvase. Sufficient material is provided for 100 amplification and cleavage reactions.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA

The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA
The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets that can be cleaved by the Guide-it Resolvase.

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Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells. 293T cells were transfected with plasmids encoding Cas9 and a sgRNA specific for the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus was generated using Terra Direct Polymerase Mix, and the PCR products were purified and cleaved using either Guide-it Resolvase or the Cel1 enzyme (Surveyor assay). Mutations were easily discernable when using the Guide-it kit. In contrast, the Surveyor Assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the ability to detect low levels of mutation.

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Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system. Panel A. Schematic of the AcGFP DNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy of AcGFP1 were transfected with 1.5 μg of plasmid DNA for Cas9 expression and 1.5 μg of a plasmid harboring one of two sgRNAs (T1 or T2) using Xfect Transfection Reagent. The cell population was assayed 6 days post-transfection for cleavage efficiency and loss of fluorescence. Panel B. Using the Guide-it Mutation Detection Kit, cleavage products were detected for both sgRNAs, indicating that both CRISPRs successfully disrupted the AcGFP1 locus. Panel C. The AcGFP1 disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS.

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The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

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631443: Guide-it Mutation Detection Kit

631443: Guide-it Mutation Detection Kit
631448 Guide-it™ Mutation Detection Kit 25 Rxns USD $237.00

The Guide-it Mutation Detection Kit contains all the reagents needed for PCR-based identification of insertions or deletions generated during cellular non-homologous end joining (NHEJ) repair. The first step is the amplification of the putative target sequence directly from cells. This kit uses Terra PCR Direct Polymerase Mix and Buffer, so there is no need to extract genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets for cleavage by the Guide-it Resolvase. Sufficient material is provided for 100 amplification and cleavage reactions.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA

The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA
The Guide-it Mutation Detection Kit is used to confirm the presence of mutations in genomic DNA. In the first step your target sequence is amplified directly from your target cells using the Terra PCR Direct Polymerase included in the kit, so there is no need to extract and purify genomic DNA from your cell population prior to amplification of your target sequence. The amplicon is then melted and hybridized to form the mismatched targets that can be cleaved by the Guide-it Resolvase.

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Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells

Comparison of the Guide-it and Surveyor assays for detecting mutations in mammalian cells. 293T cells were transfected with plasmids encoding Cas9 and a sgRNA specific for the AAVS1 locus. Transfected cells were harvested 48 hr post-transfection and mixed with untransfected cells at varying ratios. An amplicon containing the targeted AAVS1 locus was generated using Terra Direct Polymerase Mix, and the PCR products were purified and cleaved using either Guide-it Resolvase or the Cel1 enzyme (Surveyor assay). Mutations were easily discernable when using the Guide-it kit. In contrast, the Surveyor Assay showed considerable smearing, making it difficult to determine cleavage efficiency and reducing the ability to detect low levels of mutation.

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Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system

Successful knockout of AcGFP1 in HT1080 cells using the CRISPR/Cas9 system. Panel A. Schematic of the AcGFP DNA sequence and the location of sgRNAs tested and primer placement for the mutation detection assay. HT1080 cells containing a single copy of AcGFP1 were transfected with 1.5 μg of plasmid DNA for Cas9 expression and 1.5 μg of a plasmid harboring one of two sgRNAs (T1 or T2) using Xfect Transfection Reagent. The cell population was assayed 6 days post-transfection for cleavage efficiency and loss of fluorescence. Panel B. Using the Guide-it Mutation Detection Kit, cleavage products were detected for both sgRNAs, indicating that both CRISPRs successfully disrupted the AcGFP1 locus. Panel C. The AcGFP1 disruptions were functionally relevant, as a subpopulation of non-fluorescent cells could be detected by FACS.

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631448: Guide-it Mutation Detection Kit

631448: Guide-it Mutation Detection Kit
631444 Guide-it™ Indel Identification Kit 10 Rxns USD $462.00

The Guide-it Indel Identification Kit is used for characterization of insertions and deletions (indels) generated by gene editing tools, such as CRISPR/Cas9. This kit contains all of the components needed to amplify, clone, and prepare modified target sites for DNA sequence analysis. This kit uses Terra PCR Direct to amplify targets directly from crude genomic DNA extracts. The resulting pool of fragments, which may contain a variety of indels, are cloned into a prelinearized pUC19 vector using the In-Fusion cloning system. Colony PCR of individual clones using Terra PCR Direct followed by DNA sequencing allows indel characterization.

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Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting

Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting
Identification of insertions and deletions (indels) in the CD81 gene after CRISPR/Cas9 targeting. HeLa cells were transfected with plasmids encoding Cas9 and an sgRNA targeting the CD81 gene. The Guide-it Indel Identification Kit was used to prepare CD81 target sites for DNA sequence analysis. The sequencing data from six clones was aligned with the wild-type sequence, revealing a broad range of indels in the CD81 gene.

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The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions (indels) introduced by nuclease-based genome editing

The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions (indels) introduced by nuclease-based genome editing

The Guide-it Indel Identification Kit provides a complete workflow for identifying the variety of insertions and deletions (indels) introduced by nuclease-based genome editing. The protocol uses direct PCR to amplify a genomic DNA fragment (~500 to 700 bp) containing the target site directly from crude cell lysates (step 1). The resulting amplified fragments contain a pool of edited target sites from individual cells. These PCR products are cloned directly into a pre-linearized pUC19 vector using the In-Fusion Cloning system (step 2). After transformation of an optimized E. coli strain, colony PCR is used to amplify the target site from the plasmid (step 3). DNA sequencing is then used to identify the different indels generated at the targeted genomic site (step 4)

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The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells

The CRISPR/Cas9 system, a simple, RNA-programmable method to mediate genome editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA (sgRNA) directing the Cas9 endonuclease to induce a double strand break at a specific target sequence three base-pairs upstream of a PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) nonhomologous end joining, (NHEJ) resulting in gene knockout due to error-prone repair (orange), or 2) homology-directed repair (HDR), resulting in gene knockin due to the presence of a homologous repair template (purple).

631317 Xfect™ Transfection Reagent 100 Rxns USD $283.00

License Statement

ID Number  
63 Use of this product is covered by one or more of the following U.S. Patent Nos. and corresponding patent claims outside the U.S.: 6,998,115; 7,427,394. This product is intended for research purposes only. It may not be used for (i) any human or veterinary use, including without limitation therapeutic and prophylactic use, (ii) any clinical use, including without limitation diagnostic use, (iii) screening of chemical and/or biological compounds for the identification of pharmaceutically active agents (including but not limited to screening of small molecules), target validation, preclinical testing services, or drug development. Any use of this product for any of the above mentioned purposes requires a license from the Massachusetts Institute of Technology.

Xfect is a transfection reagent that creates biodegradable nanoparticles that permit superior transfection efficiency of plasmid DNA into mammalian cells. Transfections can be carried out entirely in the presence of serum.

Notice to purchaser

Our products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.

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High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent

High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent
High-efficiency transfection of seven of the most commonly used cell lines with Xfect Transfection Reagent.

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Transfection of Jurkat cells

Transfection of Jurkat cells

Transfection of Jurkat cells. Suspension cells such as Jurkat cells are notoriously very hard to transfect, but using the Xfect Transfection Reagent, you can achieve almost 30% efficiency. The transfection efficiency using the leading competitor product, on the other hand, is no higher than background.

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Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent

Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent

Xfect transfection reagent yields higher numbers of transfected, viable cells than a popular competitor reagent. The Xfect Reagent and Product L were each used according to their respective protocols to transfect HeLa cells with increasing amounts of plasmid DNA encoding the Living Colors fluorescent protein, AcGFP1. 48 hr posttransfection, AcGFP1 expression was assessed by flow cytometry in order to determine transfection efficiency and cell viability was assessed by trypan dye exclusion assay.

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Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent

Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent
Customer testimonial: transfection of primary rat cardiomyocytes with Xfect Transfection Reagent.

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Share without contaminating

Share without contaminating

Share without contaminating. Xfect Transfection Reagent is packaged in convenient, separable kits, so if you purchase the 100 reaction kit (2 x 50 rxns) or 300 reaction kit (3 x 100 rxns) you can share with your less-than-careful colleagues without fear.

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The simple Xfect Transfection Reagent protocol is completely serum-compatible

The simple Xfect Transfection Reagent protocol is completely serum-compatible
The simple Xfect Transfection Reagent protocol is completely serum-compatible.

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Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here

Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here
Our customers have indicated via survey that they have successfully used Xfect Transfection Reagent to transfect the cell lines listed here.

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631317: Xfect Transfection Reagent

631317: Xfect Transfection Reagent


Related gene editing links

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