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  • ‹ Back to Gene editing in hiPS cells
  • Tagging an endogenous gene with AcGFP1 in hiPS cells
  • Tagging an endogenous gene with a myc tag in hiPS cells
  • Generating clonal hiPS cell lines deficient in CD81
  • Introducing a tyrosinemia-related SNP in hiPS cells
  • Inserting an expression cassette into the AAVS1 locus in hiPS cells
  • Editing hiPS cells using electroporation
  • Editing hiPS cells using gesicle technology
  • Single-cell cloning of hiPS cells
Home › Learning centers › Stem cell research › Technical notes › Gene editing in hiPS cells › Editing hiPS cells using gesicle technology

Technical notes

  • Pluripotent stem cells
    • Using the DEF-CS system to culture human iPS cells
    • Comparison of the Cellartis DEF-CS system with other vendors' human iPS cell culture systems
    • Reprogramming PBMCs
    • Reprogramming fibroblasts
  • Gene editing in hiPS cells
    • Tagging an endogenous gene with AcGFP1 in hiPS cells
    • Tagging an endogenous gene with a myc tag in hiPS cells
    • Generating clonal hiPS cell lines deficient in CD81
    • Introducing a tyrosinemia-related SNP in hiPS cells
    • Inserting an expression cassette into the AAVS1 locus in hiPS cells
    • Editing hiPS cells using electroporation
    • Editing hiPS cells using gesicle technology
    • Single-cell cloning of hiPS cells
  • Organoids
    • Liver organoid differentiation from iPSCs for prediction of drug-induced liver injury
    • Generation of embryonic organoids using NDiff 227 neural differentiation medium
  • Beta cells
    • Beta cells for disease modeling
  • Hepatocytes
    • hiPS-HEP cells for disease modeling
    • hiPS-HEP cells for drug metabolism studies
    • Power medium for long-term human primary hepatocyte culture
    • iPS cell to hepatocyte differentiation system
  • Cardiomyocytes
    • Making engineered heart tissue with cardiomyocytes
  • Neural stem cells
    • RHB-A neural stem cell medium
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Tech Note

Editing hiPS cells using gesicle technology

  • Highly efficient gene editing in hiPS cells
    Gesicles deliver Cas9 protein and sgRNA, avoiding genomic integration and reducing off-target effects
  • Maintenance of pluripotency after editing, single-cell cloning, and expansion
    hiPS cells maintain high levels (>90%) of pluripotency markers Oct‑4, TRA‑1‑60, and SSEA-4
  • Stable karyotype from start to finish
    hiPS cells maintain a normal and stable karyotype throughout editing, single-cell cloning, and expansion
Introduction Results Conclusions Methods References

Introduction  

CRISPR/Cas9 is a revolutionary tool that offers simplicity and flexibility for gene editing experiments. Stem cells are ideal candidates for editing, as they are highly renewable and expandable, and they can differentiate into multiple cell types. Human induced pluripotent stem (hiPS) cells offer the greatest utility due to their practically unlimited expansion and differentiation potential.

The sequential processes of somatic cell reprogramming to create patient-specific hiPS cells, CRISPR/Cas9 gene editing, and single-cell cloning uniquely enable researchers to study how a specific genetic modification can influence function. To create cell models for the discovery of disease etiology, progression, and treatment, isogenic cell lines can be created from healthy or sick individuals, then compared (Figure 1).

Human iPS cells offer a renewable source of clonal diseased or healthy cells

Figure 1. Human iPS cells offer a renewable source of clonal diseased or healthy cells.

Cells from a healthy individual can be reprogrammed to create an expandable population of healthy hiPS cells. This healthy hiPS cell population can be edited to insert a known or theorized mutation and then expanded clonally to yield a complementary cell line that only differs from the healthy cells by the introduced mutation. Later, this edited clonal cell line can be differentiated into a cell type relevant to the disease under study (e.g., neurons, as depicted in Figure 1). Alternatively, cells from a sick individual can be reprogrammed and expanded into a diseased hiPS cell line, and then gene editing can be used to correct the mutation thought to cause the disease. This corrected clonal cell line can also be differentiated into the cell type of interest. Both of these renewable sources of diseased and healthy cell models, controlled for genetic variability, can be used for a variety of downstream applications to study and treat disease.

Challenges of editing hiPS cells and novel solutions

Despite the power and utility of CRISPR/Cas9 as an editing tool, some challenges must be overcome, particularly when editing hiPS cells. Delivery of Cas9 and the target-specific sgRNA requires an efficient method with low toxicity. Off-targt effects should be minimized by the experimental design: choose a good sgRNA with the fewest possible off-target effects, control the amount of Cas9 introduced into the cells, and limit the time that Cas9 is present in the cells. Once edited, single cells with the desired mutation need to be expanded clonally, an inefficient process. Throughout all stages of editing and single-cell-expansion experiments, hiPS cells need to be highly proliferative and pluripotent.

To address the editing challenges, we have developed a system that is based on the delivery of recombinant Cas9 protein complexed with a sgRNA targeting the gene of interest (Cas9/sgRNA ribonucleoproteins [RNPs]) via cell-derived nanovesicles, called gesicles. Gesicles are released from the plasma membrane of mammalian producer cells and can carry any cargo, such as proteins and protein complexes. CRISPR/Cas9 gesicles are generated by the coexpression of Cas9 protein, a customer-designed sgRNA, and other proteins that stimulate gesicles to be released from the producer cell membrane. Once gesicles have been made, they can be harvested, concentrated, and applied to hiPS cells, where the active Cas9/sgRNA complex is released and transported to the nucleus for efficient gene editing. The use of gesicles allows a footprint-free editing method, wherein the dose of Cas9/sgRNA RNPs and their presence in the cell is limited, reducing off-target effects as compared to plasmid-based methods.

After gesicle-based delivery of the editing machinery into hiPS cells, cells must survive the process of single-cell cloning—a major bottleneck to obtaining clones with the modification of interest. Delicate hiPS cells typically grow in colony- and/or feeder-dependent conditions, and isolating them as single cells removes survival and growth signals, reducing their viability. Promoting survival and proliferation at the single-cell level is critical for expansion of any clonal colonies containing the mutation of interest generated by the gene editing process.

To overcome the challenges with editing and single-cell cloning of hiPS cells, we have developed a method that combines the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit and the Guide-it CRISPR/Cas9 Gesicle Production System. This method utilizes the DEF-CS culture system, recognized for its suitability for genome engineering (Valton et al. 2014) and single-cell cloning (Feng et al. 2014), to promote reliable growth of hiPS cells in a feeder-free and defined environment.

With this system, hiPS cells are grown as a homogeneous monolayer and are enzymatically passaged as single cells that maintain pluripotency with a stable karyotype for more than 20 passages (Asplund et al. 2016). The same reagents are used for monolayer and single-cell culture, reducing variability. When plated as single cells in wells of a 96-well plate, at least 50% of those seeded cells will proliferate into clonal colonies. In the following experiments, we demonstrate that the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit combined with Guide-it CRISPR/Cas9 Gesicle Production System is ideal for performing footprint-free gene editing and single-cell cloning of edited hiPS cells.

Results  

Maintenance of pluripotency after editing: AcGFP1 knockout (KO) test case

An initial proof-of-concept experiment was performed to confirm that editing hiPS cells using gesicles carrying Cas9-sgRNA RNPs does not influence pluripotency (Figure 2, Panel A). First, cells from Cellartis Human iPS Cell Line 22 (ChiPSC22) were modified to stably express AcGFP1, and then these cells were cultured using the DEF-CS culture system in a 48-well plate. Next, cells were treated with 30 µl of gesicles (produced using the Guide-it CRISPR/Cas9 Gesicle Production System) containing RNP complexes with sgRNA targeting AcGFP1. The cells were then grown for nine days according to the Cellartis DEF-CS 500 Culture System User Manual. After, cells were analyzed by flow cytometry or immunocytochemistry for AcGFP1 and pluripotency markers. Untreated cells (negative control) were grown in parallel for the same amount of time as treated cells.

To determine pluripotency and AcGFP1 knockout efficiency in gesicle-treated cells, cells were labeled with a fluorescently labeled antibody specific to the pluripotency marker SSEA-4. Cells were analyzed via flow cytometry and the percentages of cells that were AcGFP1 positive and SSEA-4 positive were quantified (Figure 2, Panel B). The negative control hiPS cells were ~95% AcGFP1 positive and over 99% SSEA-4 positive, indicating the cells were unedited and pluripotent. Treatment of hiPS cells with gesicles carrying Cas9-sgRNA RNP complexes targeting AcGFP1 resulted in knockout of AcGFP1 expression in 80% of the cells. Furthermore, pluripotency—as determined by SSEA-4 expression—was maintained in 99% of analyzed cells.

Additional confirmation of knockout and pluripotency was performed using immunocytochemistry (Figure 2, Panel C). Control and gesicle-treated hiPS cells were assessed for AcGFP1 expression (green), labeled with an antibody specific to pluripotency marker Oct-4, visualized with a fluorescent-labeled secondary antibody (red), and nuclear-labeled with DAPI (blue). An isotype control for the secondary antibody was used as a negative control in both samples. In accordance with the flow cytometry data, expression of AcGFP1 in gesicle-treated cells was knocked out in the majority of cells, despite AcGFP1 being expressed in nearly all control cells. Nearly all hiPS cells were Oct-4 positive, supporting the conclusion that editing using gesicles does not alter pluripotency.

Gesicle-mediated knockout of AcGFP1 in hiPS cells does not alter pluripotency

Figure 2. Gesicle-mediated knockout of AcGFP1 hiPS cells does not alter pluripotency when performed in the DEF-CS culture system. Panel A. Gesicle-based delivery of Cas9-sgRNA RNPs was used to knock out AcGFP1 in the Cellartis Human iPS Cell Line 22 stably expressing AcGFP1. Cells were cultured under non-differentiating conditions using the Cellartis DEF-CS 500 Culture System. After editing, cells were analyzed for AcGFP1 expression and pluripotency via flow cytometry (Panel B) and immunocytochemistry (Panel C). As a negative control, untreated parental cells were grown in parallel for the same amount of time. The quantification of AcGFP1 expression in the edited cells revealed an 80% knockout. Furthermore, pluripotency of the parental and edited cell populations was maintained, with nearly 100% of cells expressing SSEA-4 (assessed via flow cytometry in Panel B) and Oct-4 (assessed via immunohistochemistry in Panel C).

Workflow for generating clonal hiPS cell lines deficient in CD81

After concluding from our proof-of-concept experiment that a successful gesicle-based knockout of a virally integrated gene can be achieved, we chose to target CD81, an endogenous membrane glycoprotein that forms complexes with integrins and plays a critical role in the infection process that leads to hepatitis C (Figure 3).

Workflow for targeted knockout of CD81 using gesicle technology

Figure 3. Workflow for targeted knockout of CD81, an endogenous gene in hiPS cells, using the Cellartis iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System. hiPS cells can be cultured, edited, and clonally expanded using the Cellartis iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System. Before and after using this system, hiPS cells are grown in the DEF-CS culture system, which maintains cells as a karyotypically stable and pluripotent monolayer.

Knockout of CD81 in cells from Cellartis Human iPS Cell Line 18 (ChiPSC18) was performed according to the Guide-it CRISPR/Cas9 Gesicle Production System user manual. 30 µl of gesicles containing Cas9 in an RNP complex with sgRNA specifically targeting CD81 was added to the cells. Flow cytometry analysis of the total population was performed seven days later to determine the CD81 KO efficiency and overall pluripotency levels. Flow cytometry was employed to isolate the sorted population of CD81-negative cells. Single cells from this sorted population were then seeded into each well of a 96-well plate by limiting dilution. Single hiPS cells were expanded into clonal lines using the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit and characterized for pluripotency and karyotype.

Successful editing of human iPS cells and maintenance of pluripotency

After gesicle treatment, assessment of CD81 expression by flow cytometry identified that 57.8% of gesicle-treated hiPS cells were CD81 negative (Figure 4, Panel A). We also interrogated the population of sorted, CD81-negative cells for the expression of pluripotency markers (Figure 4, Panel B) and identified that successfully edited hiPS cells were 92.6% Oct-4 positive, 99.7% TRA-1-60 positive, and 99.99% SSEA-4 positive. Taken together, these data indicate that hiPS cells expanded in the DEF-CS culture system can be successfully edited using gesicles while retaining their pluripotency.

CD81 negative hiPS cells retain pluripotency

Figure 4. CD81-negative hiPS cells remain pluripotent in the DEF-CS culture system. Panel A. Gesicle-based delivery of Cas9-sgRNA RNPs was used to knock out CD81 in ChiPSC18 cells. Cells were cultured under non-differentiating conditions using the Cellartis DEF-CS 500 Culture System. After editing, cells were analyzed for CD81 expression via flow cytometry. As a negative control, unedited parental cells were grown in parallel for the same amount of time. Quantification of CD81 expression in the gesicle-treated cells revealed a 57.8% knockout efficiency. Panel B. CD81-negative cells were examined for pluripotency markers Oct-4, TRA-1-60, and SSEA-4. Pluripotency of parental (negative control) and edited (CD81–) cell populations was maintained, with 92.6% of edited cells expressing Oct-4, 99.7% of edited cells expressing TRA-1-60, and ~100% of edited cells expressing SSEA-4.

Following gesicle treatment, CD81-negative hiPS cells sorted via flow cytometry were counted using a hemocytometer. Limiting dilution was performed to achieve a final theoretical concentration of 30 cells/9,600 µl. Next, 100 µl of the final cell suspension was added to each well of a 96-well plate (reaching a final theoretical concentration of 0.3 cells/well). This increased the likelihood of obtaining wells containing only single cells and minimized the existence of doublets, which prevent the derivation of a clonal population. Following two weeks of culture, we identified 20 emerging colonies, yielding a calculated single-cell survival efficiency of ~67% (Table I). These data demonstrate that the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit combined with Guide-it CRISPR/Cas9 Gesicle Production System enables highly efficient survival of single hiPS cells.

Table I. Highly efficient survival of edited clones grown in the Cellartis iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System.

Single-cell cloning with the DEF-CS system
Theoretical number of single cells plated Number of emerging colonies from single cells at 2 weeks Calculated single-cell survival
30 20 67%

Typical success rates for the generation of clonal colonies from a single-cell cloning experiment range from 1–5%. Using the DEF-CS cell culture component of the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit, we achieved an unparalleled survival rate of 67%.

Maintenance of pluripotency in expanded hiPS cell clones

Twelve of the emerging colonies from above were selected and expanded according to the user manual for the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit. Once scaled up, each individual clonal line was assessed for pluripotency and knockout of CD81 using flow cytometry (Figure 5). All clones expressed high levels of Oct-4 (88–98% positive), TRA-1-60 (97–99% positive), and SSEA-4 (98–99% positive). Moreover, all lines were found to be CD81 deficient. These data show that pluripotency markers in the expanded clones are maintained at levels comparable to those in the parental line, ChiPSC18. Thus, using the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit combined with Guide-it CRISPR/Cas9 Gesicle Production System, we successfully knocked out endogenous CD81 from a starting line and generated 12 new, edited lines that were still pluripotent.

Edited hiPS cell clones maintain pluripotency

Figure 5. Pluripotency was maintained in edited hiPS cell clones that were seeded as single cells. Individual, edited (CD81 knockout) hiPS cells were expanded into clonal lines and analyzed for expression of CD81 and three pluripotency markers via flow cytometry using antibodies against CD81, Oct-4, TRA-1-60, and SSEA-4. The parental hiPS cell line, ChiPSC18, was used as a control. As expected, all edited clones exhibited the loss of CD81 expression. Pluripotency was maintained in all edited clonal lines, as evidenced by the persistent expression of the three pluripotency markers.

Occurrence of a diverse set of indels in hiPS cell clones from the CD81 knockout experiment

We next examined the specific base-pair (bp) insertions and deletions (indels) created during the CRISPR/Cas9 editing process in the clones using the Guide-it Indel Identification Kit. Because Cas9-induced double-strand breaks are mainly repaired via the error-prone, nonhomologous end joining (NHEJ) DNA repair pathway, every cell that is edited should have a unique set of indels at the targeted gene—in this experiment, CD81. Accordingly, we saw a wide range of indels in the different clonal cell lines (Figure 6 and Table II). In some cases, CD81 knockout was accomplished via relatively small indels. For example, Clone #5 had only a 10-bp insertion/4-bp deletion on one allele and a 1-bp insertion on the other allele at the editing site. Conversely, some indels were much larger; Clone #3 had an 18-bp deletion on one allele and a 228-bp insertion on the other allele. Intriguingly, the sequence of the 228-bp insertion corresponded to a homologous sequence on chromosome 4, indicating that a different chromosome may have been used as a repair template. Taken together, these data demonstrate the diversity of indels created by CRISPR/Cas9 editing and highlight the utility in creating and screening multiple clones to account for this variability.

Diversity of indels created at the CD81 target site in edited hiPS clones

Figure 6. Graphical representation of indels created at the CD81 target site in different CRISPR/Cas9-edited hiPS cell clones. The genomic CD81 region of each clone was PCR amplified and sequenced using the Guide-it Indel Identification Kit to obtain detailed sequence information. The results show that each clone has a unique set of biallelic indels that knock out CD81. See Table II for a tabular representation of these data.

Table II. Tabular representation of the sizes and locations of indels created at the target site in CD81 for different CRISPR/Cas9-edited hiPS cell clones.

Diversity of indels in CRISPR/Cas9 gesicle-treated hiPS cell clones
Clone # 1 2 3 4 5 6 7 8 9 10 11 12
Indels (bp) Allele 1 3/
–10
–32 +228 (chr4) –39 +10/
–4
–24 –24 –36 +25/
–262
–11 +63 (chr4)/
–3
+181
Allele 2 –25 –32 –18 +169 (chr8) +1 +2/
–26
–24 +4/
–17
–7 –533 +63 (chr4)/
–3
+181

Results show that each clone has a unique set of indels that knock out CD81. For each allele, “+” values indicate the number of inserted nucleotides, while “−” values indicate the number of deleted nucleotides. If the insertion corresponds to another part of the genome, that chromosome is shown between parentheses.

Normal karyotypes observed in edited hiPS cells

CRISPR/Cas9-mediated editing and single-cell cloning can be harsh on hiPS cells. Traditionally, these processes can force selective pressures that favor unintended mutations to the karyotype, conferring competitive advantages to in vitro growth. These karyotypic abnormalities render the cells unsuitable for study. Consequently, it is essential that the karyotype remains unaltered for multiple passages after editing.

To confirm karyotype stability, we examined four of the clonal lines, each of which had been expanded from one single cell to a confluent line in a 10-cm dish over a period of approximately one month (Figure 7). All lines were found to have normal, stable karyotypes. Thus, these data show that the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit combined with Guide-it CRISPR/Cas9 Gesicle Production System effectively edits and expands single-cell hiPS cell clones without introducing karyotypic abnormalities.

Edited hiPS cell clones maintain a stable karyotype

Figure 7. hiPS cell clones that have been edited via gesicles maintain a stable karyotype after editing and clonal expansion. The karyotypes of five edited clonal cell lines were analyzed. All clonal lines showed the expected 46, XY karyotype of the original ChiPSC18 cell line. Clonal cell line #8 is shown as an example.

Conclusions  

Combining the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit with the Guide-it CRISPR/Cas9 Gesicle Production System provides an efficient and effective method to generate clonal lines of edited hiPS cells. This system can enable high editing efficiency, with no discernible effect on hiPS cell health or pluripotency. Single-cell seeding of edited hiPS cells using this combined method resulted in high survival of pluripotent, edited clones, yielding a diverse set of edited clonal lines. Critically, the expanded lines maintain a normal karyotype, rendering the cells suitable for further investigation and use in screening and disease modeling.

Methods  

Cell culture

Cellartis human iPS cells from the ChiPSC22 line (Figure 2) and the ChiPSC18 line (Figures 3–7 and Table I) were grown in the Cellartis DEF‑CS 500 Culture System before editing. Gesicles containing Cas9-sgRNA complexes were produced, harvested, and used to edit ChiPSC22 cells using the Guide-it CRISPR/Cas9 Gesicle Production System. After single-cell cloning and expansion into 48-well plates using the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit, clonal colonies were returned to the Cellartis DEF‑CS 500 Culture System for further expansion prior to indel analysis (Figure 6 and Table II) and karyotyping (Figure 7). Please refer to the Cellartis DEF‑CS 500 Culture System User Manual, the Guide-it CRISPR/Cas9 Gesicle Production System User Manual and the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit User Manual for specific culture conditions and protocols.

Flow cytometry

Labeling of Oct‑4 was performed by cell fixation and permeabilization followed by an incubation with anti-Oct‑4-PE antibody (BD Pharmingen; 20 µl for 1 x 106 cells) in PBS for 30 minutes.

Cell labeling of extracellular proteins or markers was performed following standard labeling procedures. In Figure 4, Panel A, cells were incubated for 30 minutes with anti‑SSEA‑4-PE (BD Pharmingen; 20 µl for 1 x 106 cells), anti-TRA‑1‑60-FITC (BD Pharmingen; 20 µl for 1 x 106 cells), or anti-CD81-FITC (BD Pharmingen; 20 µl for 1 x 106 cells) antibodies. In Figure 4, Panel B, cells were incubated simultaneously with anti‑SSEA‑4 and anti-TRA‑1‑60 antibodies, since they were labeled with different fluorophores (PE and FITC, respectively).

Immunocytochemistry

ChiPSC22 cells were grown in chamber slides until fixation with 4% paraformaldehyde. Cell permeabilization was achieved with 0.5% Triton X-100 for 5 minutes. After washing with PBS and blocking for 30 minutes (IHC/ICC Blocking Buffer - Low Protein; eBioscience), cells were incubated with anti-Oct‑4 antibody (diluted 1:150; eBioscience) or an IgG2a K isotype control (diluted 1:150; eBioscience) for one hour. After the incubation period, cells were washed and mounted with an antifading reagent containing DAPI (VECTASHIELD Antifade Mounting Medium with DAPI; Vector Laboratories).

Indel analysis

Indels within each edited clonal cell line were determined using the Guide-it Indel Identification Kit, following the kit′s user manual.

Karyotyping

Analysis of the karyotypes of ChiPSC18 cell lines was performed by Cell Guidance Systems.

References  

Asplund, A. et al. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev. Reports 12, 90–104 (2016).

Feng, Q. et al. Scalable Generation of Universal Platelets from Human Induced Pluripotent Stem Cells. Stem Cell Reports 3, 817–831 (2014).

Valton, J. et al. Efficient strategies for TALEN-mediated genome editing in mammalian cell lines. Methods 69, 151–170 (2014).

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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.
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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.

Y30010 Cellartis® DEF-CS™ 500 Culture System 1 Kit USD $546.00

License Statement

ID Number  
C001 This product is manufactured and sold by Takara Bio Europe AB based on a commercial license to certain intellectual property rights held by Wisconsin Alumni Research Foundation (“WARF”). This product is covered by one or more claims of U.S. Patent No. 7,514,260 and its foreign counterparts. The purchase of this product conveys to the buyer the non-transferable right to use the product for its intended use, strictly limited to purchaser’s own internal research. No other express or implied license is granted to the purchaser. Purchaser cannot have any right to use this product or its components in humans for any purposes including but not limited to diagnostics and/or therapeutics, or otherwise clinical trials. Purchase does not include any right to resell or transfer this product to a third party regardless of whether or not compensation is received. Purchasers wishing to use this product for purposes other than internal research use should contact us.

Cellartis DEF-CS 500 Culture System is a defined culture system for efficient expansion of undifferentiated human pluripotent stem cells. This kit includes basal medium, coating substrate, and additives.

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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Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System

Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System
Expansion potential of a characterized working bank of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System. The Cellartis DEF-CS Culture System can produce 2 x 109 human iPS cells within 4 passages (18–20 days) from frozen cells (2.0–2.5 x 106 cells).

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Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System

Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System
Robust growth of human induced pluripotent stem (iPS) cells in the Cellartis DEF-CS Culture System. The number of iPS cells was quantified after being cultured for three weeks using either the Cellartis DEF-CS Culture System, a reference feeder system, or four other stem cell culture systems.

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Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency

Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency
Human induced pluripotent stem cells (iPS) cells grown in the Cellartis DEF-CS Culture System have the highest proportion and intensity of markers of pluripotency. Quantitative analysis of TRA1-60 (Panel A) and SSEA4 (Panel B) expression was performed on human iPS cells after five weeks culture in either the Cellartis DEF-CS Culture System, a reference feeder cell containing system, or four different stem cell culture systems.

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Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques

Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques
Human iPS cells grown in the Cellartis DEF-CS Culture System look different from those grown with traditional aggregate culture techniques. Freshly passaged human iPS cells were cultured for 5 days in either the Cellartis DEF-CS Culture System, on feeder cells, in mTeSR 1 medium (STEMCELL Technologies), or in Essential 8 Medium (E8; Life Technologies).

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Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype

Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype
Human induced pluripotent stem (iPS) cells cultured long-term in the Cellartis DEF-CS Culture System retain a normal karyotype. The human iPS cell line ChiPSC18 was cultured for 20 passages in the Cellartis DEF-CS Culture System. Chromosomal analysis indicates that the cells retain a normal karyotype.

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Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System

Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System

Human induced pluripotent stem (iPS) cells can be passaged as single cells in the Cellartis DEF-CS Culture System. A single GFP-actin iPS cell was isolated and placed in the well of a culture dish. Twenty-four hours after seeding, morphology was assessed by fluorescence microscopy at 20x (Panel A) and 40x (Panel B) magnification. Sixteen days later, the single GFP-actin iPS cell had proliferated into numerous cells as evidenced by microscopic observation at 4x (Panel C), 10x (Panel D), 20x (Panel E), and 40x (Panel F) magnification.

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Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System

Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System

Human pluripotent stem cells remain undifferentiated when cultured in the Cellartis DEF-CS Culture System. Human iPS cells cultured for 23 passages in the Cellartis DEF-CS Culture System were characterized by Oct-4 staining (Panel A) and nuclear staining (Panel B).

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Y30010: Cellartis DEF-CS 500 Culture System

Y30010: Cellartis DEF-CS 500 Culture System

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That's GOOD Science!

What does it take to generate good science? Careful planning, dedicated researchers, and the right tools. At Takara Bio, we thoughtfully develop best-in-class products to tackle your most challenging research problems, and have an expert team of technical support professionals to help you along the way, all at superior value.

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Takara Bio USA, Inc. provides kits, reagents, instruments, and services that help researchers explore questions about gene discovery, regulation, and function. As a member of the Takara Bio Group, Takara Bio USA is part of a company that holds a leadership position in the global market and is committed to improving the human condition through biotechnology. Our mission is to develop high-quality innovative tools and services to accelerate discovery.

FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES (EXCEPT AS SPECIFICALLY NOTED).

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