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  • ‹ Back to CRISPR-Cas9
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Expression of AcGFP1-tagged tubulin in iPS cells Efficient gene knockins in iPS cells using ssDNA
Blog post about Marson paper Blog post: CRISPR takes a giant step towards the clinic
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Expression of AcGFP1-tagged tubulin in iPS cells Efficient gene knockins in iPS cells using ssDNA
Blog post about Marson paper Blog post: CRISPR takes a giant step towards the clinic

Long ssDNA for knockins

Long ssDNA for gene knockin experiments

CRISPR/Cas9 and other gene editing tools have been successfully used for obtaining knockout mutations, but knockin mutations have proven to be much harder to introduce. A major challenge has been the production and delivery of the repair template along with the Cas9-sgRNA ribonucleoprotein complex.

CRISPR/Cas9 and other gene editing tools have been successfully used for obtaining knockout mutations, but knockin mutations have proven to be much harder to introduce. A major challenge has been the production and delivery of the repair template along with the Cas9-sgRNA ribonucleoprotein complex.

Although single-stranded DNA (ssDNA) repair templates have recently been shown to have several advantages over double-stranded DNA (dsDNA) templates, the usefulness of long ssDNA templates is limited due to the difficulty and cost of producing them. The Guide-it Long ssDNA Strandase Kit is designed to produce a long ssDNA oligo of up to 5 kb in length for use as a repair template in knockin experiments using CRISPR/Cas9 or other gene editing tools. This kit provides a simple method for converting a dsDNA PCR product into ssDNA by selectively digesting either the sense or the antisense strand. The kit contains sufficient reagents to create 50 ssDNA strands (25 pairs of sense and antisense strands).

Benefits of using ssDNA as a template over dsDNA:

  • Drastically reduced tendency to randomly integrate into the genome, resulting in an improved gene editing efficiency
  • Low cytotoxic response to ssDNA template delivery
  • No expression from nonintegrated templates, making identification of correctly edited clones significantly easier
  • NOTE: The Guide-it Long ssDNA Production System includes a NucleoSpin Gel and PCR Clean-up kit to purify the strandase reaction before using it for gene editing
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Cat. # Product Size Price License Quantity Details
632644 Guide-it™ Long ssDNA Production System 25 Rxns $469.00

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™ Long ssDNA Production System product License Agreement can be found by clicking here.
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The Guide-it Long ssDNA Production System enables production of single-stranded DNA (ssDNA) oligos up to 5 kb in length for use as repair templates in knockin experiments involving CRISPR/Cas9 or other genome editing tools. Included with the system are sufficient reagents for production of 50 different ssDNA oligos (25 pairs of sense and antisense ssDNAs), and the NucleoSpin Gel and PCR Clean-up kit for purification of ssDNA prior to target-cell delivery.

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

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FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci. Cas9-sgRNA RNPs targeting SEC61B and TUBA1A and corresponding AcGFP1 ssDNA homology-directed repair (HDR) templates generated using the Guide-it Long ssDNA Production System were electroporated into ChiPSC18 cells. Following culturing, electroporated target cells and non-electroporated negative control cells were analyzed for GFP expression by FACS. Using this approach, knockin efficiencies of 12.55% and 2.75% were observed for insertion of AcGFP1 at SEC61B and TUBA1A, respectively.

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PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS. ChiPSC18 cell populations were electroporated with Cas9-sgRNA RNPs and sense or antisense ssDNA HDR templates (labeled ssDNA-1 or ssDNA-2, respectively) that were designed for knockin of AcGFP1 at the SEC61B locus. The cells were sorted on the basis of GFP expression via FACS. Individual clones from the GFP-positive populations were isolated by limiting dilution and cultured to yield clonal cell populations. Genomic DNA extracted from the clonal populations was analyzed by PCR using primers (L-F and R-R, respectively) designed to anneal on either side of the GFP insertion site as shown in the schematic. The GFP and homology arm sequences included in the ssDNA templates are shown in green and light blue, respectively, while the genomic sequences outside the region targeted by the ssDNA homology arms are shown in dark blue. The PCR primers were designed so that the presence of the inserted AcGFP1 sequence yielded a 1.885-kb PCR product, while absence of the AcGFP1 insert yielded a 1.159-kb PCR product. Gel electrophoresis of PCR products confirmed the occurrence of monoallelic knockins of AcGFP1 at SEC61B, represented by double bands (ssDNA-1 Lane C5, and ssDNA-2 Lane C15), and biallelic knockins of AcGFP1 at SEC61B, represented by single bands (ssDNA-1 Lanes C1, C7, C8, C11, and ssDNA-2 Lanes C2, C6, C8, and C10). Two clones lacking any copies of the AcGFP1 insert were also identified (ssDNA-1 Lane C10, and ssDNA-2 Lane C11).

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Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence immediately 5' relative to Exon 1 of SEC61B. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-SEC61B fusion protein would be driven by the endogenous SEC61B promoter. Binding sites for PCR primers used to detect the inserted AcGFP1 sequence are shown by blue arrows.

Back

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence at the 5' end of Exon 2 of TUBA1A. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-TUBA1A fusion protein would be driven by the endogenous TUBA1A promoter.

Back

Toxicity comparison for electroporation of dsDNA or ssDNA

Toxicity comparison for electroporation of dsDNA or ssDNA

Toxicity comparison for electroporation of dsDNA or ssDNA. ChiPSC18 cells were electroporated with Cas9-sgRNA RNPs targeting the TUBA1A locus, in the absence or presence of homology-directed repair (HDR) templates consisting of dsDNA or ssDNA. After 48 hours of culturing, imaging was performed to compare the toxicity associated with each HDR template. While electroporation of either DNA template resulted in increased toxicity compared to electroporation of Cas9-sgRNA RNPs alone (as evidenced by lower cell counts following culturing), application of the ssDNA template resulted in greatly reduced toxicity compared to the dsDNA template.

Back

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates. ChiPSC18 cells were edited using Cas9-sgRNAs and ssDNA HDR templates designed for knockin of AcGFP1 at the SEC61B and TUBA1A loci, respectively. GFP-positive clonal cell lines obtained from each edited population were then stained with DAPI and analyzed by fluorescence microscopy. Expression of GFP-SEC61B was observed in endosomal compartments (left), while GFP-TUBA1A localized to microtubules (right).

632645 Guide-it™ Long ssDNA Strandase Kit 25 Rxns $362.00

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™ Long ssDNA Strandase Kit product License Agreement can be found by clicking here.
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

The Guide-it Long ssDNA Strandase Kit enables production of single-stranded DNA (ssDNA) oligos up to 5 kb in length for use as repair templates in knockin experiments involving CRISPR/Cas9 or other genome editing tools. The kit includes sufficient reagents for production of 50 different ssDNA oligos (25 pairs of sense and antisense ssDNAs). In contrast with the Guide-it Long ssDNA Production System (Cat. # 632644), this kit does not include a clean-up kit for purifying ssDNA prior to delivery to target cells.

Notice to purchaser

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

Documents Components You May Also Like Image Data

Back

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci

FACS analysis of AcGFP1 knockin efficiencies at the SEC61B and TUBA1A loci. Cas9-sgRNA RNPs targeting SEC61B and TUBA1A and corresponding AcGFP1 ssDNA homology-directed repair (HDR) templates generated using the Guide-it Long ssDNA Production System were electroporated into ChiPSC18 cells. Following culturing, electroporated target cells and non-electroporated negative control cells were analyzed for GFP expression by FACS. Using this approach, knockin efficiencies of 12.55% and 2.75% were observed for insertion of AcGFP1 at SEC61B and TUBA1A, respectively.

Back

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS

PCR analysis of AcGFP1 knockin at the SEC61B locus in clonal populations isolated by FACS. ChiPSC18 cell populations were electroporated with Cas9-sgRNA RNPs and sense or antisense ssDNA HDR templates (labeled ssDNA-1 or ssDNA-2, respectively) that were designed for knockin of AcGFP1 at the SEC61B locus. The cells were sorted on the basis of GFP expression via FACS. Individual clones from the GFP-positive populations were isolated by limiting dilution and cultured to yield clonal cell populations. Genomic DNA extracted from the clonal populations was analyzed by PCR using primers (L-F and R-R, respectively) designed to anneal on either side of the GFP insertion site as shown in the schematic. The GFP and homology arm sequences included in the ssDNA templates are shown in green and light blue, respectively, while the genomic sequences outside the region targeted by the ssDNA homology arms are shown in dark blue. The PCR primers were designed so that the presence of the inserted AcGFP1 sequence yielded a 1.885-kb PCR product, while absence of the AcGFP1 insert yielded a 1.159-kb PCR product. Gel electrophoresis of PCR products confirmed the occurrence of monoallelic knockins of AcGFP1 at SEC61B, represented by double bands (ssDNA-1 Lane C5, and ssDNA-2 Lane C15), and biallelic knockins of AcGFP1 at SEC61B, represented by single bands (ssDNA-1 Lanes C1, C7, C8, C11, and ssDNA-2 Lanes C2, C6, C8, and C10). Two clones lacking any copies of the AcGFP1 insert were also identified (ssDNA-1 Lane C10, and ssDNA-2 Lane C11).

Back

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus

Schematic depicting Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the SEC61B locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence immediately 5' relative to Exon 1 of SEC61B. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-SEC61B fusion protein would be driven by the endogenous SEC61B promoter. Binding sites for PCR primers used to detect the inserted AcGFP1 sequence are shown by blue arrows.

Back

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus

Schematic of Cas9-sgRNA cleavage site and ssDNA construct designed for knockin of AcGFP1 at the TUBA1A locus. The ssDNA construct generated using the Guide-it Long ssDNA Production System included the AcGFP1 coding sequence (~700 nt in length) flanked on either side by homology arms ~350 nt in length. The homology arms were designed to insert the AcGFP1 sequence at the 5' end of Exon 2 of TUBA1A. As indicated, the ssDNA construct did not include a promoter sequence, so that expression of the resulting AcGFP1-TUBA1A fusion protein would be driven by the endogenous TUBA1A promoter.

Back

Toxicity comparison for electroporation of dsDNA or ssDNA

Toxicity comparison for electroporation of dsDNA or ssDNA

Toxicity comparison for electroporation of dsDNA or ssDNA. ChiPSC18 cells were electroporated with Cas9-sgRNA RNPs targeting the TUBA1A locus, in the absence or presence of homology-directed repair (HDR) templates consisting of dsDNA or ssDNA. After 48 hours of culturing, imaging was performed to compare the toxicity associated with each HDR template. While electroporation of either DNA template resulted in increased toxicity compared to electroporation of Cas9-sgRNA RNPs alone (as evidenced by lower cell counts following culturing), application of the ssDNA template resulted in greatly reduced toxicity compared to the dsDNA template.

Back

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates

In vivo expression of AcGFP1 fusion proteins in cells edited using Cas9-sgRNA RNPs and ssDNA HDR templates. ChiPSC18 cells were edited using Cas9-sgRNAs and ssDNA HDR templates designed for knockin of AcGFP1 at the SEC61B and TUBA1A loci, respectively. GFP-positive clonal cell lines obtained from each edited population were then stained with DAPI and analyzed by fluorescence microscopy. Expression of GFP-SEC61B was observed in endosomal compartments (left), while GFP-TUBA1A localized to microtubules (right).

HDR FAQs Homology-directed repair FAQs
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Gene editing tools Genome editing overview

Overview

  • Create long ssDNA donor templates up to 5 kb in length
  • Fast, easy-to-use, three-step protocol
  • No random integration or expression, leading to improved editing efficiency
  • ssDNA donor templates are far less toxic to cells than dsDNA templates

More Information

Applications

  • Donor templates for knockin experiments requiring DNA fragments >200 bp

Additional product information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


CRISPR/Cas9 information

Choosing sgRNA design tools

Browse a collection of sgRNA design tools for Cas9-based genome editing experiments.

Choosing a target sequence for CRISPR/Cas9 gene editing

Learn how to design sgRNA sequences for successful gene editing.

The CRISPR/Cas9 system for targeted genome editing

Overview of CRISPR/Cas9 system for genome editing.

CRISPR/Cas9 genome editing tools

An overview of tools available for each step in a successful genome editing workflow.

Gene editing technical notes

Delivery of Cas9 and sgRNA to mammalian cells using a variety of innovative tools.

CRISPR SNP detection webinar

Watch our webinar and learn how you can easily screen hundreds of clonal cells for SNPs.

CRISPR/Cas9 gesicles overview

Learn about Guide-it CRISPR/Cas9 Gesicle Production System components and workflow.

CRISPR library screening webinar

Watch this webinar to learn how you can perform genome-wide lentiviral sgRNA screens easily.

Guide-it SNP Screening Kit FAQs

Get answers to frequently asked questions and view a video explaining the enzymatic assay.

Choosing an HDR template format

Watch a webinar on how to choose the right HDR template for knockin experiments.


Gene editing resources

Product finder

Use this gene editing product finder to quickly locate kits for screening, delivery, and downstream assays.

CRISPR tools and information

What is CRISPR/Cas9? Need help designing the best guide RNAs? Learn all this and more.

Genome-wide library screening

Learn how to conduct a CRISPR/Cas9 guide RNA library phenotypic screen and view data demonstrating the use of our library.

CRISPR/Cas9 knockouts

Technotes and tools used to create or study CRISPR-Cas9-mediated gene knockouts (indels).

CRISPR/Cas9 knockins

Information, technotes and tools used to create or study gene knockouts by CRISPR/Cas9 and HDR.

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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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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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