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632612: pGuide-it-sgRNA1 Vector System

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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. 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. 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. (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 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. 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. 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. 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. 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. 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. 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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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. 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

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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. 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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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. 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. 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.

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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.

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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. 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. 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. 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 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

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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. 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. 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. 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

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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. 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 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 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).

Back

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.

Back

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.

Back

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.

Back

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

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

Back

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.

Back

The simple Xfect Transfection Reagent protocol is completely serum-compatible

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