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Home › Learning centers › Gene function › Gene editing › CRISPR/Cas9 knockouts › Screening for effective guide RNAs

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In vitro transcription and screening Kits In vitro sgRNA transcription and screening kits
Tech Note

In vitro cleavage efficiency of sgRNAs correlates with functional genome editing in target cells

  • A novel in vitro assay to test sgRNA cleavage efficiency
    Screen various sgRNAs to determine the most effective sgRNAs prior to delivering to your cells
  • Accurate prediction of sgRNA cleavage efficiency
    sgRNA cleavage efficiency predicted in vitro correlates with in vivo cleavage as assessed by both a nuclease assay and functional analysis
Introduction Results Conclusions References

Introduction  

In CRISPR/Cas9 genome editing, targeting the Cas9 nuclease to a specific genomic locus is solely mediated by a user-defined sgRNA. Currently available web-based tools for sgRNA design will return a variety of candidate sgRNAs for a single gene target. Despite these in silico predictions, not every sgRNA will exhibit equivalent cleavage efficiency. Given this inconsistency, it is necessary to screen multiple sgRNAs to identify the most effective one.

Results  

An in vitro assay to test sgRNA cleavage efficiency

The Guide-it sgRNA Screening Kit is a complete system for predicting the cleavage efficacy of sgRNAs in vitro, prior to use for genome editing in cells (Figure 1). With this kit, a template containing a sgRNA-target site is created by PCR; then the test sgRNA and recombinant Cas9 nuclease are added. The efficiency of Cas9-mediated cleavage can be measured by agarose gel electrophoresis.

Guide-it sgRNA Screening Kit workflow

Figure 1. Overview of the Guide-it sgRNA Screening Kit protocol. A PCR amplicon containing a sgRNA target site is synthesized from genomic DNA (Step 1). The PCR fragment is then combined with a candidate sgRNA and recombinant Cas9 (Step 2). The entire reaction is separated by agarose gel electrophoresis (Step 3). Since the sgRNA-target sequence is located asymmetrically within the amplicon, cleavage by the Cas9/sgRNA complex results in two bands of unequal length that can be easily distinguished on an agarose gel.

sgRNAs exhibit different cleavage efficiencies

CRISPR/Cas9 genome editing was used to disrupt the CXCR4 locus in HeLa cells. CXCR4 encodes a cell surface chemokine receptor that interacts with the CXCL12 chemokine and plays an important role in the immune system. In this experiment, four different sgRNAs targeting the CXCR4 locus were tested using the Guide-it sgRNA Screening Kit. Briefly, sgRNAs targeting the CXCR4 gene were synthesized using the Guide-it sgRNA In Vitro Transcription Kit. A PCR fragment containing the sgRNA target sequence was mixed with recombinant Cas9 protein and each sgRNA. The cleavage reaction was analyzed by agarose gel electrophoresis. Densitometry (Cong et al., 2013) showed that sgRNA3 had the lowest cleavage efficiency (Figure 2).

In vitro sgRNA cleavage efficiency

Figure 2. Differences in in vitro cleavage efficiency as determined by the Guide-it sgRNA Screening Kit. The cleavage efficiency of four different sgRNAs targeting the CXCR4 locus were tested. A PCR fragment containing the CXCR4 target sequence was synthesized and mixed with Cas9 and each sgRNA. A negative control that lacked sgRNA was included for comparison (NC). Cleavage efficiency was assessed by agarose gel electrophoresis and measured using densitometry (%).

In vitro cleavage efficiency predicts in vivo cleavage

HeLa cells were cotransfected with plasmids encoding Cas9 and each of the four different sgRNAs tested above. The presence of mutations in the CXCR4 locus as was assayed using the Guide-it Mutation Detection Kit. This assay uses a mismatch-specific nuclease, Guide-it Resolvase, to identify insertions or deletions in specific loci in cells treated with engineered nucleases. Mismatches were detected with high efficiency in cells treated with sgRNAs 1, 2, and 4 (Figure 3). However, cells treated with sgRNA3 exhibited a very low efficiency of mismatches, consistent with the efficiency predicted by the Guide-it sgRNA Screening Kit (Figure 2).

Mutation detection

Figure 3. sgRNA-mediated cleavage in HeLa cells as determined by the Mutation Detection Kit. HeLa cells were co-transfected with plasmids encoding Cas9 and one of the four different sgRNAs using Xfect Transfection Reagent. Six days after transfection, cells were assayed for the presence of mutations using Guide-it Resolvase, a mismatch-specific nuclease. Cleavage fragments were present for all sgRNAs except sgRNA3, indicating low Cas9 guiding efficiency for this particular sgRNA.

CXCR4 gene disruption was also assessed by flow cytometry; since CXCR4 is a cell surface receptor, it can be detected by flow cytometry using a FITC-labeled CXCR4 antibody. Disruption in CXCR4 expression could be detected in cells transfected with Cas9 and sgRNAs 1, 2, and 4 (Figure 4). In contrast, for cells transfected with Cas9 and sgRNA3, a much smaller proportion of the cells had disruption of CXCR4 expression. These functional data confirm the results obtained by both the Guide-it sgRNA Screening Kit and the Guide-it Mutation Detection Kit.

Functional knockout of CXCR4

Figure 4. Flow cytometric analysis detects sgRNA-mediated loss of CXCR4 function. Knockout of the CXCR4 gene by CRISPR/Cas9 editing results in in reduced protein expression; therefore, FITC staining is inversely correlated with efficient genome editing. In this experiment, HeLa cells were cotransfected with plasmids encoding Cas9 and each of four sgRNAs, and then stained with a FITC-labeled antibody against CXCR4. The percentage (%) of the cell population that was not labeled with FITC is shown in blue. Cells treated with Cas9 and sgRNA3 exhibited the greatest percentage of FITC+ cells and the least efficient genome editing.

Conclusions  

There is a clear correlation between in vitro sgRNA cleavage efficiency as predicted by the Guide-it sgRNA Screening Kit and in vivo sgRNA-mediated cleavage as assessed by the presence of indels and functional gene knockout (Figure 5). These results indicate that the Guide-it sgRNA Screening Kit is an ideal method for screening for ineffective sgRNAs during CRISPR/Cas9 genome editing projects.

Correlation between in vitro cleavage efficiency and gene knockout

Figure 5. The Guide-it sgRNA Screening Kit accurately predicts in vivo sgRNA efficacy. Cleavage efficiency was assessed by in vitro cleavage (Figure 2) and the Guide-it Mutation Detection Kit (Figure 3); functional knockout was assessed by flow cytometry (Figure 4, % of CXCR4− cells). There is a clear correlation between the efficiency predicted by the Guide-it sgRNA Screening Kit, the estimation of in vivo cleavage provided by the Mutation Detection Kit, and the level of functional knockout (via flow cytometry).

References  

Cong, L. et al. (2013) Multiplex genome engineering using CRISPR/Cas9 systems. Science 339(6121):819–23.

Related Products

Cat. # Product Size Price License Quantity Details
632636 Guide-it™ Complete sgRNA Screening System 50 Rxns $848.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
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. A copy of the Guide-it™ Complete sgRNA Screening System product License Agreement can be found by clicking here.

The Guide-it Complete sgRNA Screening System includes everything needed for the simple production, cleanup, and evaluation of single guide RNAs (sgRNAs) for CRISPR/Cas9 studies, including PCR reagents for amplifying your genomic target and recombinant Cas9 for in vitro analysis of the transcribed sgRNA.

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.

Documents Components Image Data

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Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
632638 Guide-it™ IVT RNA Clean-Up Kit 50 Rxns $335.00

The Guide-it IVT RNA Clean-Up Kit is included as part of the Guide-it In Vitro Transcription Kit and is used to purify sgRNA transcribed using the Guide-it In Vitro Transcription Kit.

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.

Documents Components Image Data

Back

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
631443 Guide-it™ Mutation Detection Kit 100 Rxns $449.00

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.

Documents Components You May Also Like Image Data Resources

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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.

Back

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.

Back

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).

631448 Guide-it™ Mutation Detection Kit 25 Rxns $203.00

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.

Documents Components You May Also Like Image Data

Back

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.

Back

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.

Back

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.

Back

Guide-it Mutation Detection Kit Product Photo: 25 Rxns (631448)

Guide-it Mutation Detection Kit Product Photo: 25 Rxns (631448)
632635 Guide-it™ sgRNA In Vitro Transcription Kit 50 Rxns $695.00

License Statement

ID Number  
M54 This product is covered by the claims of U.S. Patent Nos. 7,704,713 and its foreign counterparts. 
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. A copy of the Guide-it™ sgRNA In Vitro Transcription Kit product License Agreement can be found by clicking here.

The Guide-it In Vitro Transcription Kit contains the necessary reagents for the transcription and cleanup of your sgRNA sequences. It is combined with the Guide-it sgRNA Screening Kit as part of the Guide-it Complete sgRNA screening system which can be used for the simple production, cleanup, and evaluation of single guide RNAs (sgRNAs) for CRISPR/Cas9 studies.

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.

Documents Components Image Data

Back

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.
632639 Guide-it™ sgRNA Screening Kit 50 Rxns $375.00

The Guide-it sgRNA Screening Kit contains PCR reagents to amplify genomic DNA from your target cells and recombinant Cas9 for the in vitro screening of your sgRNA to help you pick the best guide sequences before moving to experiments in living cells or animals. It is combined with the Guide-it In VItro Transcription kit as part of the Guide-it Complete sgRNA Screening System.

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.

Documents Components Image Data

Back

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein

Production of sgRNA by in vitro transcription and testing for cleavage using recombinant Cas9 protein
Schematic highlighting the key steps for using Guide-it kits for the production of high amounts of single guide RNA (sgRNA) and screening for target site cleavage efficacy using recombinant Cas9 protein.

Back

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System

sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System
sgRNA yield and quality for different gene targets using the Guide-it Complete sgRNA Screening System. Panel A. 20 µl of each sgRNA in vitro transcription (IVT) reaction was incubated for 4 hr at 37°C. The yield for each transcribed sgRNA was quantified using a NanoDrop spectrophotometer. Panel B. Each sgRNA IVT reaction was a run on an Agilent Bioanalyzer to assay for sample quality.

Back

sgRNA quality produced via in vitro transcription

sgRNA quality produced via in vitro transcription
sgRNA quality produced via in vitro transcription. sgRNAs produced using the Guide-it sgRNA In Vitro Transcription Kit were compared with sgRNAs produced using a competitor’s kit. The Guide-it kit produced a high-quality, single band for each reaction, while the competitor’s kit showed unwanted byproducts.

Back

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs

Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs
Flow chart describing how the Guide-it sgRNA In Vitro Transcription Kit (including the Guide-it IVT RNA Clean-Up Kit) and Guide-it sgRNA Screening Kit work together in the Guide-it Complete sgRNA Screening System, which can be used to synthesize and test the efficacy of sgRNAs.

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Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, 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, TBUSA 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.

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Capturem Trypsin for a rapid, efficient mass spectometry workflow at room temperature.

Speed up your mass spec workflow

Capturem Trypsin provides rapid, efficient, and complete digestion of protein samples, allowing an uninterrupted mass spectometry workflow at room temperature for downstream protein analysis. This product utilizes our novel Capturem technology in a spin column format with membrane-immobilized trypsin. Capturem Trypsin Columns may be used to completely digest protein samples in less than a minute with digestion efficiencies (protein coverage) comparable to or better than those obtained using in-solution trypsin digestion.

Capturem trypsin technology

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Mapping the brain, one cell type at a time

Learn about pioneering efforts to map the mammalian brain using single-cell transcriptomics.

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Takara Bio USA, Inc. (TBUSA, formerly known as Clontech Laboratories, 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, TBUSA 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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