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.
Highlights
CRISPR-Cas[9] based gene editing has become the method of choice for rapid generation of cell lines and transgenic animals with targeted genomic alterations, thereby greatly facilitating genetic engineering for understanding gene and protein function
We demonstrate that homoduplex and heteroduplex DNA fragments carrying small indels display differences in migration when subjected to 4–6% agarose gel electrophoresis
PCR products of the target region were directly sequenced (Supplementary Table 1), and DNA of representative wild-type (WT) and all 10 different mutants identified was size-separated by electrophoresis on 4% agarose gels (Fig. 1 and Supplementary Figs S1 and S2)
Summary
CRISPR-Cas[9] based gene editing has become the method of choice for rapid generation of cell lines and transgenic animals with targeted genomic alterations, thereby greatly facilitating genetic engineering for understanding gene and protein function. CRISPR (clustered regularly interspaced short palindromic repeats) gene editing uses the endonuclease Cas[9] combined with a guide RNA (gRNA) designed to cleave a targeted genomic sequence and induce a double-strand DNA break[1,2]. We present an alternative simple two-step heteroduplex analysis method involving PCR of genomic DNA and agarose gel electrophoresis to genotype tissue or cells with CRISPR induced small allelic indels. We have implemented it for three applications: (i) identify genetically altered founder pups from CRISPR-Cas[9] mediated targeting of exon 10 of the mouse Adgrb[3] gene, (ii) genotype the www.nature.com/scientificreports/. The Scn8a gene encodes a protein that forms the ion pore region of the alpha subunit of voltage gated sodium channel Nav1.6, which is widely expressed in neurons[13]
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