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  • ‹ Back to Oligo design tool for SNP screening
  • Oligo design tool user guide (SNPs)
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Oligo design tool for assaying SNPs Oligo design tool for assaying SNPs
Home › Learning centers › Gene function › Gene editing › Creating and screening for SNPs › Oligo design tool for SNP screening › Oligo design tool user guide (SNPs)

Creating and screening for SNPs

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Oligo design tool for assaying SNPs Oligo design tool for assaying SNPs
User guide

Using the Guide-it Knockin Screening Kit oligo design tool for SNP detection

The Guide-it SNP oligo design tool is a web-based tool that can be used to design probes and control oligos for use in SNP detection assays performed with the Guide-it Knockin Screening Kit. To obtain assay-specific oligo sequences in a ready-to-order format, simply input a pair of genomic target sequences corresponding to the two alleles you wish to screen (e.g., sequences encoding wild-type (WT) or SNP bases at the site of interest). A guide for using this tool is provided below.

NOTE: This tool designs oligo probes to screen for single-base substitutions only, not for insertions.

  • To design oligos for the detection of multiple substitutions in the genomic target region, please refer to the section below, "Detecting multiple edits".
  • To design oligos for the detection of targeted insertions, please refer to the Guide-it Knockin Screening Kit User Manual.
Inputs Outputs Detecting multiple edits Support

Inputs  

The oligo design tool takes as input two sequences that correspond to the two alleles to be screened (e.g., wild-type/unedited and SNP/edited versions of the genomic target sequence), with each including at least 35 bases on either side of the modification site (i.e., the site at which a researcher is seeking to engineer a single-nucleotide substitution using genome editing technology). The first nucleotide sequence input typically corresponds to the native or wild-type version of the genomic target sequence (i.e., the sequence before editing), while the second nucleotide sequence is identical to the first sequence, except that it includes a single-nucleotide substitution (i.e., the sequence after editing). Once the sequences have been input successfully, clicking the [Submit] button will initiate the oligo design process. The [Reset] button can be used to clear the inputs if needed. An example of this input is shown below:

Oligo design tool input

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

  • Edited input sequences must include exactly one SNP.
  • Both wild-type and edited input sequences must be the same length (e.g., if one is 512 bases, the other is also 512).
  • Input sequence lengths cannot exceed 1,000 bases.
  • Input sequences should consist of at least 35 bases on either side of the SNP site. For optimal probe design, we recommend including ≥50 bases on either side.

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.

To design oligos to detect multiple substitutions in parallel, please refer to the "Detecting multiple edits" section.

Outputs  

Following a successful submission, the oligo design tool will generate a sequence alignment and the recommended sequences for probe and control oligos. Users can return to the input screen by refreshing the page or clicking the [Back] button of the web browser.

Alignment of input sequences

The inputs are first validated to ensure they meet all the requirements mentioned above. Alignment of the sequences is generated with the position of the SNP indicated using standard annotation. An example of this step is shown below:

Oligo design alignment

Probe and control oligos

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

  • Displacement oligo (presented below in purple with the noncomplementary base indicated by underline)
  • Flap-probe oligo - SNP (presented below in green with the 5' fixed sequence indicated by underline and the hexanediol modification indicated by "3C6" in black font)
  • Flap-probe oligo - WT (presented below in orange with the 5' fixed sequence indicated by underline and the hexanediol modification indicated by "3C6" in black font)
  • SNP control oligo (presented below in blue with the targeted base indicated in red)
  • Wild-type control oligo (presented below in blue-green with the targeted base indicated in red)

Depending on the input sequences, the tool will design oligos according to either Design 1 or Design 2 (targeting either the sense or the antisense strand of the genomic target sequence). At the bottom of each output, the tool will indicate which design was employed and present a color-coded diagram indicating the orientations for each design. 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.

Oligo design outputs

Detecting multiple edits  

SNP screening assays performed with the Guide-it Knockin Screening Kit can be used to detect the presence of more than one edit in the genomic target region because complete hybridization is required between the assay probe oligos and PCR product to generate a fluorescent signal.

Our oligo design tool for SNP screening assays only allows for the inclusion of one SNP (one nucleotide difference) between the input sequences. However, it can still be used to design assay probes for scenarios in which additional edits are incorporated in the genomic target region (e.g., scenarios when the user wants to engineer multiple nucleotide substitutions in parallel to generate an amino acid substitution or mutate a PAM, etc.). Below we provide an example of how to design assay probes for such scenarios.

Designing an assay to detect two adjacent nucleotide substitutions

Here we demonstrate how to use the oligo design tool to generate assay probes for detecting two adjacent nucleotide substitutions, using the example of an M164W mutation (ATG>TGG) in the PSEN gene:

PSEN gene with two adjacent nucleotied substitutions

For this editing scenario, the first base edit starting from the 5' end of the genomic target sequence will be used as a reference to generate the displacement and flap-probe oligos. Here, it would be the A > T substitution (lowercase "t" indicated by the box):

The first base edit from the 5' end is 't'

  1. Generate the SNP flap-probe oligo and SNP control oligo for detection of the edited sequence and the displacement oligo:

    1. In the oligo design tool, include the first base edit (A > T substitution, dark blue) in only the second input sequence ("Sequence after editing")
    2. Include the second base edit (T > G substitution, light blue) in both of the input sequences (for both "Sequence before editing" and "Sequence after editing", the input base should be 'g')
    3. Click [Submit] to have the tool design the probes and control oligo
    4. From the output, use the sequences for the displacement oligo, SNP flap-probe oligo, and SNP control oligo to generate oligos
    5. Do not use the sequences for the WT flap-probe oligo or WT control oligo
    Oligo tool results for the first step
  1. Generate the WT flap-probe oligo and WT control oligo for detection of the unedited sequence:

    1. In the oligo design tool, include the first base edit (A > T substitution, dark blue) in only the second input sequence ("Sequence after editing")
    2. Exclude the second base edit (T > G substitution) from both of the input sequences (for both "Sequence before editing" and "Sequence after editing", the input base should be 'T')
    3. Click [Submit] to have the tool design the probe and control oligo
    4. From the output, use the sequences for the WT flap-probe oligo and WT control oligo to generate oligos
    5. Do not use the sequences for the displacement oligo, SNP flap-probe oligo, or SNP control oligo
    Oligo tool results after the second step

Support  

While we have attempted to make using 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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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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