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  • ‹ Back to Oligo design tool for detecting precise insertions
  • Oligo design tool user guide (insertions)
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Home › Learning centers › Gene function › Gene editing › CRISPR/Cas9 knockins › Oligo design tool for detecting precise insertions › Oligo design tool user guide (insertions)

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    • Oligo design tool user guide (insertions)
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User guide

Using the Guide-it Knockin Screening Kit oligo design tool for detection of precise insertions

The Guide-it oligo design tool for precise insertions is a web-based tool that can be used to design probes and control oligos to perform insertion detection assays with the Guide-it Knockin Screening Kit. To obtain assay-specific oligo sequences in a ready-to-order format, simply input sequences corresponding to the junctions of the wild-type genomic target locus and the insert that will result from editing. A guide for using this tool is provided below.

NOTE: This tool designs oligo probes to screen for insertions longer than one nucleotide.

To design oligos for the detection of single-base substitutions in the genomic target region, please use the Guide-it SNP oligo design tool.

Inputs Outputs Technical support

Inputs  

Each insert screening assay performed using the Guide-it Knockin Screening Kit involves a set of oligos designed to detect seamless, error-free incorporation of a designated insert sequence at a specific genomic target site. You will need to input (in the 5'→3' orientation) four sequence fragments that correspond to the 5' and 3' junctions of the insert and genomic target site following successful editing. (Please see figure below for further clarification.)

  • To generate the oligos to detect the 5' junction:
    • Enter the wild-type sequence 5' upstream of the insertion (1)
    • Enter the 5' terminal sequence of the insert (2)
  • To generate the oligos to detect the 3' junction:
    • Enter the 3' terminal sequence of the insert (3)
    • Enter the wild-type sequence 3' downstream of the insertion (4)

Please note that the following conditions must be satisfied in order to use the tool successfully:

  • Input sequences cannot exceed 1,000 bases
  • Input sequences should consist of at least 35 bases

NOTE: Please be careful to avoid the inclusion of extra spaces when inputting sequences, as these will be interpreted as part of the input and will likely return an error message.

Outputs  

Following submission of the four sequences, the oligo design tool will output displacement, flap-probe, wild-type control, and knockin control oligos for analysis of both the 5' and 3' junctions.

Users can return to the input screen by refreshing the page or clicking the [Back] button of the web browser.

Generation of the final edited 5' and 3' junctions

The inputs are first validated to ensure they meet all the requirements mentioned above. The top of the results page will display the sequences of both 5' and 3' junctions with the insert sequences depicted in lowercase. The user can check if the introduced sequences are correct. An example of this output is shown below:

Probe and control oligos

Sequences of the following four pairs of oligos are provided in a ready-to-order format:

  • 2 x Displacement oligos (presented below in purple with the noncomplementary base indicated by underline)
  • 2 x Flap-probe oligos (presented below in green for the 5' junction and orange for the 3' junction, with the 5' fixed sequences indicated by underline and the hexanediol modifications indicated by "3C6" in black font)
  • 2 x Knockin control oligos (presented in blue with the insert sequence indicated in dark blue and in lowercase)
  • 2 x Wild-type control oligos (presented in blue)

Depending on the input sequences, the tool will design oligos according to either Design 1 or Design 2. At the bottom of each output, the tool will indicate which design was employed and present a color-coded diagram indicating the orientations of the design at the 5' and 3' junctions. For more detailed information about the oligo design process for the Guide-it Knockin Screening Kit, please refer to the product user manual. For more insights about the Guide-it Knockin Screening Kit assay, please refer to our FAQs page.

Technical support  

While we have attempted to make the oligo design tool as simple and straightforward as possible, users may run into difficulties that are technical or biological in nature. In either case, please reach out to our technical support team for assistance with your designs. Providing additional information such as any error messages encountered, and snapshots of the corresponding inputs and outputs will be extremely helpful in these instances. For all support questions, please contact a Technical Support Scientist through our support page or the Live Chat option on the bottom right corner of our web pages.

Related products

Cat. # Product Size License Quantity Details
632659 Guide-it™ Knockin Screening Kit 100 Rxns USD $434.00

License Statement

ID Number  
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

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

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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

632659: Guide-it Knockin Screening Kit

632659: Guide-it Knockin Screening Kit
632660 Guide-it™ Knockin Screening Kit 400 Rxns USD $1112.00

License Statement

ID Number  
325 Patent pending. For further information, please contact a Takara Bio USA licensing representative by email at licensing@takarabio.com.

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

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

632660: Guide-it Knockin Screening Kit

632660: Guide-it Knockin Screening Kit

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That's GOOD Science!

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

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

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

Clontech, TaKaRa, cellartis

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