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Tech note: efficient SNP engineering Tech note: efficient SNP screening
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Tech note: efficient SNP engineering Tech note: efficient SNP screening

Guide-it Knockin Screening Kit

Screening for precise edits

A common bottleneck for genome editing applications involves a lack of efficient methods for detecting successfully engineered SNPs or insertions. While many researchers rely on sequencing to verify editing, this typically requires isolation and expansion of single-cell clones and yields data that can be challenging to analyze. The Guide-it Knockin Screening Kit provides a streamlined method for identifying precise edits at any locus, either in heterogeneous or clonal cell populations. The assay takes only 4 hours to complete and can be performed using basic laboratory equipment.

A common bottleneck for genome editing applications involves a lack of efficient methods for detecting successfully engineered SNPs or insertions. While many researchers rely on sequencing to verify editing, this typically requires isolation and expansion of single-cell clones and yields data that can be challenging to analyze. The Guide-it Knockin Screening Kit provides a streamlined method for identifying precise edits at any locus, either in heterogeneous or clonal cell populations. The assay takes only 4 hours to complete and can be performed using basic laboratory equipment.

One of the most powerful applications of genome editing technologies such as the CRISPR/Cas system is the ability to introduce precise changes at genomic loci of interest. However, the likelihood of success for this type of application is generally low because it often relies on an endogenous repair mechanism known as homology-directed repair (HDR) that occurs at relatively low frequency. Consequently, there are two different stages when the detection of successful HDR events is critically important. The first stage involves optimization of experimental conditions to achieve the highest percentage of error-free HDR events in an edited population before moving forward with the isolation of single-cell clones. The second stage involves identification of cell lines carrying the edit of interest after single-cell isolation and expansion in 96-well plates. The Guide-it Knockin Screening Kit addresses the need for sensitive detection of successful HDR at both stages by providing a simple fluorescence-based method that can be applied to screen edited populations as well as clones from 96-well plates for edits ranging in size from single-nucleotide substitutions to longer insertions.

The kit assay consists of PCR amplification of the genomic target site, followed by an enzymatic assay employing assay-specific oligos that can be designed with our online tool. The dual-color fluorescence-based readout from the assay can be measured using a standard plate reader or qPCR machine. A positive fluorescent signal from the assay is highly correlated with the correct introduction of the desired edit. For engineering SNPs, the assay enables detection of single-nucleotide substitutions with high sensitivity in both mixed and clonal populations and can be used to positively identify heterozygous clones carrying one copy each of the edited (SNP) and unedited (WT) alleles. For scenarios involving knockin of longer sequences, the assay allows for the simultaneous detection of seamless insertions at both 5' and 3' ends of the incorporated sequence.

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Cat. # Product Size Price License Quantity Details
632660 Guide-it™ Knockin Screening Kit 400 Rxns USD $1218.00

License Statement

ID Number  
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
440 This Product is protected by one or more patents from the family comprising: US 2019/0218544 and any corresponding patents, divisionals, continuations, patent application and foreign filings sharing priority with the same family.

The Guide-it Knockin Screening Kit enables sensitive detection of successful homologous recombination (HR) events in mixed or clonal cell populations edited using technologies such as the CRISPR/Cas9 system. The kit employs a simple fluorescence-based method that can reliably detect successful HR events regardless of the knockin length (from single-nucleotide substitutions to longer insertions) or the sequence of the genomic region surrounding the edit. The simple and rapid kit workflow consists of PCR amplification of the genomic target site followed by an enzymatic assay with green and red fluorescent readouts. The enzymatic assay employs a standard fluorescence plate reader or qPCR machine for endpoint detection of fluorescence, and no additional special instrumentation is required. The overall workflow takes approximately four hours to complete, and the stringency of the assay is such that detection of fluorescent signal(s) positively correlates with the presence of the desired sequence at the genomic target site. For research applications that involve engineering SNPs, the assay can be used to positively identify heterozygous clones carrying one copy each of two different alleles (e.g., SNP and WT alleles). For scenarios involving knockin of longer sequences, the assay allows for the simultaneous detection of seamless insertions at both 5' and 3' ends of the incorporated sequence.

Cat. # 632660 includes sufficient quantities of reagents for performing 400 assays.

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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Simultaneous detection of WT and SNP alleles carrying a silent PAM mutation at the PSEN1 locus in a bulk-edited iPS cell population

Simultaneous detection of WT and SNP alleles carrying a silent PAM mutation at the PSEN1 locus in a bulk-edited iPS cell population

To demonstrate the SNP-detection capabilities of the Guide-it Knockin Screening Kit, we used CRISPR/Cas editing technology to generate an iPS cell line heterozygous for a variant of the PSEN1 gene encoding an A>G substitution (M164V) associated with early-onset Alzheimer's disease. Panel A. HDR templates carrying silent PAM mutations used to perform precise editing at the PSEN1 locus. Following successful HDR, the PSEN1 locus will encode either a WT or SNP allele combined with a silent mutation in the neighboring PAM sequence. Panel B. Design of displacement and flap-probe oligos for detection of edits involving silent mutation of the PAM sequence (G>C) and introduction of the SNP (A>G). The displacement oligo probe (in purple) is designed to hybridize in a similar manner when either allele is present (HDR wt silent or HDR M164V). The two different flap-probe oligos (in orange and green) are designed to fully hybridize to either the HDR wt silent allele encoding the PAM mutation (generating a red signal) or the HDR M164V allele encoding both the PAM mutation and the SNP (generating a green signal), respectively. The fixed sequences responsible for generating the fluorescent signals are underlined for each flap-probe oligo. Assayed bases for each allele are indicated in lowercase font.

Back

Common outcomes when engineering SNPs

Common outcomes when engineering SNPs

An example of a single-nucleotide edit (G>T) is shown. Panel A. Outcomes at the genomic target site. When cleavage fails to occur at the target site or is followed by accurate, nonhomologous end joining (NHEJ)-based repair, the result is the wild-type (WT) sequence. When cleavage is followed by inaccurate NHEJ-based repair, the result is an insertion or deletion (Indel) at the target site possibly causing a knockout (KO, a highly probable outcome). When cleavage is followed by accurate HDR, a SNP is introduced at the target site. Panel B. Combined allelic outcomes in diploid cells. When editing is performed in diploid cells, the outcomes for each allele can vary, generating multiple possible combinations. Cells can remain homozygous (Wild type; top), they can have one or both alleles modified via inaccurate NHEJ (Indel; middle), or they can have one or both alleles modified with the desired SNP (Successful HDR; bottom).

Back

SNP analysis workflow for the Guide-it Knockin Screening Kit

SNP analysis workflow for the Guide-it Knockin Screening Kit

This example workflow demonstrates analysis of a G>A substitution, where G is the wild-type base edited to an A. After genome editing, single cells expanded to clonal cell lines can have several different genotypic outcomes at the genomic target site of interest. After PCR amplification of the target site, the PCR product is annealed simultaneously with different oligo probes: a displacement oligo (purple) in combination with either flap-probe oligo A (green; encoding the SNP allele, A) or flap-probe oligo B (orange; encoding the WT allele, G). After the annealing of the oligos to the PCR products, the Guide-it Flapase enzyme (indicated with scissors) recognizes a complete base pairing and cleaves the 5′ portion of the flap-probe oligo (shaded green or orange). The cleaved flaps are then detected by corresponding Guide-it flap detectors, which yield green or red fluorescent signals, respectively. In the example above, analysis of a clonal cell line that is homozygous WT (G/G) at the site of interest yields only a red signal, while analysis of a heterozygous clone carrying both edited and WT alleles (G/A) yields both red and green signals.

Back

Detection of precise editing at an endogenous locus in bulk-edited and clonal iPSC populations

Detection of precise editing at an endogenous locus in bulk-edited and clonal iPSC populations

Panel A. Editing outcomes following successful HDR at an anonymous locus of interest. Following successful HDR, the edited locus will encode either a SNP (in blue, lowercase) or a WT base (in purple) combined with a silent PAM mutation (in red, lowercase). Panel B. Detection of successful HDR in bulk-edited iPSCs. Displacement and flap-probe oligos were designed to detect WT silent or SNP alleles, yielding red and green fluorescent signals, respectively. In independent experiments, cells were electroporated with Cas9 protein alone (negative control), Cas9-sgRNA RNP complexes (KO), or RNP complexes combined with antisense SNP or SNP/WT silent ssODN mixtures. Synthetic oligos encoding the WT silent or SNP sequences were assayed in parallel as positive controls. For each editing scenario in which ssODNs were included in the electroporation mixture, successful HDR could be detected in the bulk population using the Guide-it Knockin Screening Kit, as indicated by the resulting fluorescent signals. Panel C. Detection of successful HDR in clonal cell lines. Clones obtained from single cells isolated by flow cytometry were screened for both edits (SNP and WT silent). While successful incorporation of either edit could be detected in separate clonal cell lines, no heterozygous clones carrying both edits were identified.

Back

The Guide-it Knockin Screening Kit provides a method for detecting full-length knockin insertions

The Guide-it Knockin Screening Kit provides a method for detecting full-length knockin insertions

After the genome editing event, bulk-edited population or clonal cell lines isolated via FACS or limiting dilution may carry wild-type, indel, or full-length insertions. After DNA extraction from the clonal cells and subsequent PCR amplification of the target site, the PCR product is annealed with two different sets of displacement and flap probes: one that hybridizes with the 5' end of the insert (Flap-probe oligo A; green), and the other with the 3' end (Flap-probe oligo B; orange). If the full-length HR event has been successful and seamless, the full hybridization of the probes at both termini will generate both green and red fluorescent signals after the cleavage of the respective flap probes by the Guide-it Flapase. Detection of only one signal (red or green) indicates an insertion truncated on either the 5' or 3' end, respectively. The lack of fluorescence is indicative of the presence of the wild-type sequence or an indel at the target site.

Back

Beta-tester data: successful identification of heterozygous edited clones

Beta-tester data: successful identification of heterozygous edited clones

Genotypes determined via bioinformatic analysis of the Sanger sequencing data are indicated along the X-axis (WT, wild-type; SNP, accurate HDR; Indel, NHEJ; unknown, software could not determine). The presence of edited (SNP) and wild-type (WT) alleles in the clones as determined by the Guide-it Knockin Screening Kit are demonstrated by fluorescence signal intensities indicated in blue (green fluorescence) and purple (red fluorescence), respectively. While the results of the knockin screening were consistent with the bioinformatic analysis of the Sanger sequencing traces for a majority of clones analyzed, there were several instances where the Sanger sequencing analysis missed or called some clones incorrectly.

Back

632660: Guide-it Knockin Screening Kit

632660: Guide-it Knockin Screening Kit
632659 Guide-it™ Knockin Screening Kit 100 Rxns USD $477.00

License Statement

ID Number  
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.
440 This Product is protected by one or more patents from the family comprising: US 2019/0218544 and any corresponding patents, divisionals, continuations, patent application and foreign filings sharing priority with the same family.

The Guide-it Knockin Screening Kit enables sensitive detection of successful homologous recombination (HR) events in mixed or clonal cell populations edited using technologies such as the CRISPR/Cas9 system. The kit employs a simple fluorescence-based method that can reliably detect successful HR events regardless of the knockin length (from single-nucleotide substitutions to longer insertions) or the sequence of the genomic region surrounding the edit. The simple and rapid kit workflow consists of PCR amplification of the genomic target site followed by an enzymatic assay with green and red fluorescent readouts. The enzymatic assay employs a standard fluorescence plate reader or qPCR machine for endpoint detection of fluorescence, and no additional special instrumentation is required. The overall workflow takes approximately four hours to complete, and the stringency of the assay is such that detection of fluorescent signal(s) positively correlates with the presence of the desired sequence at the genomic target site. For research applications that involve engineering SNPs, the assay can be used to positively identify heterozygous clones carrying one copy each of two different alleles (e.g., SNP and WT alleles). For scenarios involving knockin of longer sequences, the assay allows for the simultaneous detection of seamless insertions at both 5' and 3' ends of the incorporated sequence.

Cat. # 632659 includes sufficient quantities of reagents for performing 100 assays.

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

Simultaneous detection of WT and SNP alleles carrying a silent PAM mutation at the PSEN1 locus in a bulk-edited iPS cell population

Simultaneous detection of WT and SNP alleles carrying a silent PAM mutation at the PSEN1 locus in a bulk-edited iPS cell population

To demonstrate the SNP-detection capabilities of the Guide-it Knockin Screening Kit, we used CRISPR/Cas editing technology to generate an iPS cell line heterozygous for a variant of the PSEN1 gene encoding an A>G substitution (M164V) associated with early-onset Alzheimer's disease. Panel A. HDR templates carrying silent PAM mutations used to perform precise editing at the PSEN1 locus. Following successful HDR, the PSEN1 locus will encode either a WT or SNP allele combined with a silent mutation in the neighboring PAM sequence. Panel B. Design of displacement and flap-probe oligos for detection of edits involving silent mutation of the PAM sequence (G>C) and introduction of the SNP (A>G). The displacement oligo probe (in purple) is designed to hybridize in a similar manner when either allele is present (HDR wt silent or HDR M164V). The two different flap-probe oligos (in orange and green) are designed to fully hybridize to either the HDR wt silent allele encoding the PAM mutation (generating a red signal) or the HDR M164V allele encoding both the PAM mutation and the SNP (generating a green signal), respectively. The fixed sequences responsible for generating the fluorescent signals are underlined for each flap-probe oligo. Assayed bases for each allele are indicated in lowercase font.

Back

Common outcomes when engineering SNPs

Common outcomes when engineering SNPs

An example of a single-nucleotide edit (G>T) is shown. Panel A. Outcomes at the genomic target site. When cleavage fails to occur at the target site or is followed by accurate, nonhomologous end joining (NHEJ)-based repair, the result is the wild-type (WT) sequence. When cleavage is followed by inaccurate NHEJ-based repair, the result is an insertion or deletion (Indel) at the target site possibly causing a knockout (KO, a highly probable outcome). When cleavage is followed by accurate HDR, a SNP is introduced at the target site. Panel B. Combined allelic outcomes in diploid cells. When editing is performed in diploid cells, the outcomes for each allele can vary, generating multiple possible combinations. Cells can remain homozygous (Wild type; top), they can have one or both alleles modified via inaccurate NHEJ (Indel; middle), or they can have one or both alleles modified with the desired SNP (Successful HDR; bottom).

Back

SNP analysis workflow for the Guide-it Knockin Screening Kit

SNP analysis workflow for the Guide-it Knockin Screening Kit

This example workflow demonstrates analysis of a G>A substitution, where G is the wild-type base edited to an A. After genome editing, single cells expanded to clonal cell lines can have several different genotypic outcomes at the genomic target site of interest. After PCR amplification of the target site, the PCR product is annealed simultaneously with different oligo probes: a displacement oligo (purple) in combination with either flap-probe oligo A (green; encoding the SNP allele, A) or flap-probe oligo B (orange; encoding the WT allele, G). After the annealing of the oligos to the PCR products, the Guide-it Flapase enzyme (indicated with scissors) recognizes a complete base pairing and cleaves the 5′ portion of the flap-probe oligo (shaded green or orange). The cleaved flaps are then detected by corresponding Guide-it flap detectors, which yield green or red fluorescent signals, respectively. In the example above, analysis of a clonal cell line that is homozygous WT (G/G) at the site of interest yields only a red signal, while analysis of a heterozygous clone carrying both edited and WT alleles (G/A) yields both red and green signals.

Back

Detection of precise editing at an endogenous locus in bulk-edited and clonal iPSC populations

Detection of precise editing at an endogenous locus in bulk-edited and clonal iPSC populations

Panel A. Editing outcomes following successful HDR at an anonymous locus of interest. Following successful HDR, the edited locus will encode either a SNP (in blue, lowercase) or a WT base (in purple) combined with a silent PAM mutation (in red, lowercase). Panel B. Detection of successful HDR in bulk-edited iPSCs. Displacement and flap-probe oligos were designed to detect WT silent or SNP alleles, yielding red and green fluorescent signals, respectively. In independent experiments, cells were electroporated with Cas9 protein alone (negative control), Cas9-sgRNA RNP complexes (KO), or RNP complexes combined with antisense SNP or SNP/WT silent ssODN mixtures. Synthetic oligos encoding the WT silent or SNP sequences were assayed in parallel as positive controls. For each editing scenario in which ssODNs were included in the electroporation mixture, successful HDR could be detected in the bulk population using the Guide-it Knockin Screening Kit, as indicated by the resulting fluorescent signals. Panel C. Detection of successful HDR in clonal cell lines. Clones obtained from single cells isolated by flow cytometry were screened for both edits (SNP and WT silent). While successful incorporation of either edit could be detected in separate clonal cell lines, no heterozygous clones carrying both edits were identified.

Back

The Guide-it Knockin Screening Kit provides a method for detecting full-length knockin insertions

The Guide-it Knockin Screening Kit provides a method for detecting full-length knockin insertions

After the genome editing event, bulk-edited population or clonal cell lines isolated via FACS or limiting dilution may carry wild-type, indel, or full-length insertions. After DNA extraction from the clonal cells and subsequent PCR amplification of the target site, the PCR product is annealed with two different sets of displacement and flap probes: one that hybridizes with the 5' end of the insert (Flap-probe oligo A; green), and the other with the 3' end (Flap-probe oligo B; orange). If the full-length HR event has been successful and seamless, the full hybridization of the probes at both termini will generate both green and red fluorescent signals after the cleavage of the respective flap probes by the Guide-it Flapase. Detection of only one signal (red or green) indicates an insertion truncated on either the 5' or 3' end, respectively. The lack of fluorescence is indicative of the presence of the wild-type sequence or an indel at the target site.

Back

Beta-tester data: successful identification of heterozygous edited clones

Beta-tester data: successful identification of heterozygous edited clones

Genotypes determined via bioinformatic analysis of the Sanger sequencing data are indicated along the X-axis (WT, wild-type; SNP, accurate HDR; Indel, NHEJ; unknown, software could not determine). The presence of edited (SNP) and wild-type (WT) alleles in the clones as determined by the Guide-it Knockin Screening Kit are demonstrated by fluorescence signal intensities indicated in blue (green fluorescence) and purple (red fluorescence), respectively. While the results of the knockin screening were consistent with the bioinformatic analysis of the Sanger sequencing traces for a majority of clones analyzed, there were several instances where the Sanger sequencing analysis missed or called some clones incorrectly.

Back

632659: Guide-it Knockin Screening Kit

632659: Guide-it Knockin Screening Kit

Oligo design tool for assaying SNPs Oligo design tool for assaying SNPs
Tech note: efficient SNP engineering Tech note: efficient SNP screening
HDR FAQs Homology-directed repair FAQs

Overview

  • Accurate detection of precise nucleotide substitutions or insertions following genome editing
  • Can be used to assay any edit at any genomic locus
  • Suitable for analysis of heterogeneous (bulk-edited) or clonal cell populations
  • Dual-color chemistry enables simultaneous detection of two different alleles (e.g., SNP and WT) in heterozygous clones
  • Simple and rapid workflow takes just 4 hours to complete and requires only basic laboratory equipment
  • Online tool available for streamlined design of assay-specific oligos

More Information

Applications

  • Rapid detection of single-nucleotide substitutions or precise insertions in edited cell populations
  • Simultaneous detection of edited and unedited alleles in heterozygous clones
  • Analysis of editing efficiency in heterogeneous populations prior to single-cell cloning

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.


Beta-tester testimonials on the Guide-it Knockin Screening Kit

"The kit was very good at identifying WT/SNV hets, which is often what I'm looking to uncover."
—Dr. Justin McDonough, THE JACKSON LABORATORY
"The assay was easy and fairly quick to perform...Sanger sequencing was complicated to analyze since one allele was edited and the other allele had an indel resulting in mixed electropherograms...I like the fact that the fluorescent probes are built into the kit and the user only needs to supply fairly standard probes which are not very expensive."
—Dr. Karin Nitiss, UNIVERSITY OF ILLINOIS AT CHICAGO

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FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. © 2025 Takara Bio Inc. All Rights Reserved. All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at takarabio.com.

Takara Bio

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.

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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 exceptional 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.

Explore what makes good science possible

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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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