A simple genotyping method to detect small CRISPR-Cas9 induced indels by agarose gel electrophoresis

CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we devel...

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Bibliographic Details
Published in:Scientific reports 2019-03, Vol.9 (1), p.4437, Article 4437
Main Authors: Bhattacharya, Debanjan, Van Meir, Erwin G
Format: Article
Language:English
Subjects:
Alleles
Animals
CRISPR
CRISPR-Cas Systems
Denaturation
Electrophoresis, Agar Gel - methods
Electrophoretic mobility
Female
Gel electrophoresis
Gene Editing
Genome editing
Genotype
Genotypes
Genotyping
Genotyping Techniques
Hybrids
INDEL Mutation
Membrane Proteins - antagonists & inhibitors
Membrane Proteins - genetics
Membrane Proteins - metabolism
Mice
Mice, Transgenic
NAV1.6 Voltage-Gated Sodium Channel - chemistry
NAV1.6 Voltage-Gated Sodium Channel - genetics
NAV1.6 Voltage-Gated Sodium Channel - metabolism
Nerve Tissue Proteins - antagonists & inhibitors
Nerve Tissue Proteins - genetics
Nerve Tissue Proteins - metabolism
Polymerase Chain Reaction
Renaturation
Transgenic mice
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Summary:CRISPR gene editing creates indels in targeted genes that are detected by genotyping. Separating PCR products generated from wild-type versus mutant alleles with small indels based on size is beyond the resolution capacity of regular agarose gel electrophoresis. To overcome this limitation, we developed a simple genotyping method that exploits the differential electrophoretic mobility of homoduplex versus heteroduplex DNA hybrids in high concentration agarose gels. First, the CRISPR target region is PCR amplified and homo- and hetero-duplexed amplicons formed during the last annealing cycle are separated by 4-6% agarose gel electrophoresis. WT/mutant heteroduplexes migrate more slowly and are distinguished from WT or mutant homoduplexes. Heterozygous alleles are immediately identified as they produce two distinct bands, while homozygous wild-type or mutant alleles yield a single band. To discriminate the latter, equal amounts of PCR products of homozygous samples are mixed with wild-type control samples, subjected to one denaturation/renaturation cycle and products are electrophoresed again. Samples from homozygous mutant alleles now produce two bands, while those from wild-type alleles yield single bands. This method is simple, fast and inexpensive and can identify indels >2 bp. in size in founder pups and genotype offspring in established transgenic mice colonies.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-019-39950-4