Guide-itTM Knockin Screening Kit

Description

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. Our products are to be used forResearch 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. Back 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 thePSEN1gene encoding an A>G substitution M164V associated with early-onset Alzheimers disease.Panel A.HDR templates carrying silent PAM mutations used to perform precise editing at thePSEN1locus. Following successful HDR, thePSEN1locus 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 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 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 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 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 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