Guide-it Knockin Screening Kit
Guide-it Knockin Screening Kit
Brand: Takara Bio.
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Guide-it Knockin Screening Kit
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.
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
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
Detection of precise editing at the PSEN1 locus in a bulk-edited iPS cell population
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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. Detection of successful HDR in bulk-edited iPS cells. Alternative editing approaches using either sense or antisense HDR templates were employed, and the Guide-it Knockin Screening Kit was used to determine whether successful editing could be detected for each case. Displacement and flap-probe oligos were designed to detect WT or SNP alleles carrying silent (G>C) PAM mutations encoded by the corresponding HDR templates, 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 sense or antisense WT/SNP HDR template mixtures. Synthetic oligos encoding the WT or SNP sequences combined with the silent PAM mutation were assayed in parallel as positive controls. For each editing scenario in which HDR templates were included in the electroporation mixture, successful HDR could be detected in the bulk population. The lack of green and red fluorescent signals for the negative controls confirmed the specificity of the flap-probe oligos since they did not yield a false-positive fluorescent signal in the presence of the unedited WT sequence.
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).
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.
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.
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.
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.
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Detection of precise editing 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. Detection of successful HDR in bulk-edited iPS cells. Alternative editing approaches using either sense or antisense HDR templates were employed, and the Guide-it Knockin Screening Kit was used to determine whether successful editing could be detected for each case. Displacement and flap-probe oligos were designed to detect WT or SNP alleles carrying silent (G>C) PAM mutations encoded by the corresponding HDR templates, 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 sense or antisense WT/SNP HDR template mixtures. Synthetic oligos encoding the WT or SNP sequences combined with the silent PAM mutation were assayed in parallel as positive controls. For each editing scenario in which HDR templates were included in the electroporation mixture, successful HDR could be detected in the bulk population. The lack of green and red fluorescent signals for the negative controls confirmed the specificity of the flap-probe oligos since they did not yield a false-positive fluorescent signal in the presence of the unedited WT sequence.
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).
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.
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.
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.
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.
