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

• Type of Material: Article
• Authors: Debanjan Bhattacharya, Emory University; Erwin Van Meir, Emory University
• Language: English
• Date: 2019-03-14
• Publisher: Nature Research (part of Springer Nature): Fully open access journals
• Publication Version: Final Published Version
• Copyright Statement: © 2019, The Author(s).
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1038/s41598-019-39950-4
• Title of Journal or Parent Work: Scientific Reports
• ISSN: 2045-2322
• Volume: 9
• Issue: 1
• Start Page: 4437
• End Page: 4437
• Grant/Funding Information: The work was supported by grants from the NIH (R01-NS096236 and P30-CA138292) and the CURE Childhood Cancer Foundation.
• Keywords: Multidisciplinary Sciences; Science & Technology; MUTATIONS; MOUSE; Science & Technology - Other Topics
• Research Categories: Biology, Neuroscience; Health Sciences, Oncology; Biology, Genetics

Abstract

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.