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Case Study: High-throughput SNP Screening Downstream of CRISPR/Cas9 Gene Editing

One of the most powerful applications of genome editing is the introduction of nucleotide substitutions in specific genomic sites. This can be used to mimic single-nucleotide polymorphisms (SNPs) or to generate stop codons that yield precise gene knockouts. However, screening hundreds of clones for a single edited nucleotide remains a challenge, especially in the absence of a corresponding phenotype.

The Guide-it™ SNP Screening Kit, developed by Takara Bio, addresses this need and allows the screening of ninety-six samples for single-nucleotide edits in only four hours. The final fluorescent signal indicating the presence of the introduced SNP can be detected using a standard plate reader, without requiring any special instrumentation.

The Guide-it SNP Screening Kit enables detection of single-nucleotide substitutions in cells, which have been edited using technologies such as the CRISPR/Cas9 system. The fast and simple workflow comprises DNA extraction and PCR amplification of the genomic target site, followed by an enzymatic assay, using a structure-specific endonuclease that generates a fluorescent readout. This method performs comparably regardless of the nucleotide substitution being assayed, the zygosity of the clone, or the sequence of the targeted locus. The kit has been validated with mammalian cells, but the assay is not species-specific. The provided DNA extraction buffer is also suitable for plants.

The assay, employed by the Guide-it SNP Screening Kit, consists of the following steps:

  1. Extract genomic DNA from clonal cell lines using MightyPrep (25 min)
  2. PCR amplification of the target sequence (1 hr 45 min)
  3. Annealing of the PCR product and the oligo probes (25 min)
  4. Perform the Guide-it Flapase enzymatic assay
  5. Fluorescence readout using a plate reader (5 min)

After PCR amplification of the target site, the PCR product is annealed with two different complementary oligo probes, the Displacement oligo and the Flap probe, that hybridize with the DNA sample in the region adjacent to the interrogated base, defined as the actual SNP base for which you are screening. Following the annealing of the two DNA oligos with the PCR product, the kit uses a recombinant, structure-specific nuclease: Guide-it Flapase. The enzyme recognises a complete base-pairing between the Flap probe and the PCR product in the resulting three-dimensional structure and subsequently cleaves the Flap. This cleavage event causes the release of the Flap oligo, which is detected downstream by the Guide-it Flap detector, generating a fluorescence signal that can be measured using a plate reader (lower panel). Therefore, the detection of a fluorescence signal is equivalent to an exact base pairing between your Flap probe and the DNA sample.

On the other hand, Guide-it Flapase is unable to cleave gapped structures (upper panel). In case of the absence of the desired SNP, no fluorescent signal will occur.

Kit workflow

The kit workflow, depicted above, involves PCR amplification of the genomic region surrounding the site of the assayed edit (a G-to-A substitution in the example), followed by hybridization of the PCR product with two complementary, assay-specific oligos, referred to as the “displacement oligo” and “flap-probe oligo” (green and purple, respectively). Hybridization of the PCR product with the oligos forms either of these structures, depending on whether the edit has occurred (the base at the position being assayed is referred to as the “interrogated base”). Presence of the assayed edit (A) in the PCR product results in formation of a “double-flap” structure, which is recognised and cleaved by the Guide-it Flapase enzyme (a structure-specific nuclease), yielding a fluorescent signal that can be detected using a standard fluorescence plate reader. Absence of the assayed edit (G) leads to formation of a “gapped” structure, which is not cleaved by Guide-it Flapase, such that no signal is produced.

Detection of nucleotide substitutions

The Guide-it SNP Screening Kit has been successfully used to analyse SNPs at 18 different loci in the human genome, corresponding to 14 different genes. A subset of the results is shown in the graph below:

Detecting various nucleotide substitutions from genomic DNA

Genomic DNA samples, which were obtained from the Coriell Institute, which were either wild-type or homozygous for the indicated substitutions were analyzed using the Guide-it SNP Screening Kit. All substitutions were successfully detected, as demonstrated by the strong fluorescent signals obtained for samples that were homozygous (+/+, blue) for the indicated substitutions relative to signals obtained for wild-type (–/–, orange) and negative control (NT, gray) samples.

Suitable for genotyping applications

Genotyping can be performed by designing separate flap-probe oligos for each nucleotide to be assayed at a given target site and performing independent assays, as demonstrated below:

Using the Guide-it SNP Screening Kit for genotyping

Samples from the Coriell Institute carrying the indicated SNP at the NCP1 locus were analyzed using the Guide-it SNP Screening Kit (top) and by Sanger sequencing (bottom). The analysis determined which samples were homozygous or heterozygous for an A>G substitution by employing two flap-probe oligos in independent assays designed to detect A (blue graphs) or G (gray graphs).

The assay performs well regardless of whether extra PCR products are generated during amplification of the genomic target sequence. Included in the image below are examples of PCR products that have been successfully used to detect nucleotide substitutions with this kit:

PCR products visible in each gel lane were successfully analysed for nucleotide substitutions involving the indicated genomic loci. As indicated by the gel, the size of the amplified target can range between 200 bp and 700 bp. For SNP detection assays involving the DBT(E2) and MTHFR gene sequences, amplicons of two different sizes (“s” and “l”) were successfully used.

Takara Bio Europe is part of the Takara Bio Group, a leading supplier of tools for life scientists worldwide, developing innovative technologies in Next-Generation Sequencing, Gene Delivery, Gene Function, and much more.

Find out more about the era of targeted breeding at the qPCR strand at the 4Bio Summit: Europe. See the agenda here.

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