Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection
| Autor: | Jordan A. Shavit, R. Kc, David T. Burke, Jodi M. Wilkowski, Anshika Srivastava, Stephanie L. Bielas, Catherine E. Richter |
|---|---|
| Rok vydání: | 2016 |
| Předmět: |
0106 biological sciences
0301 basic medicine DNA End-Joining Repair Genotyping Techniques Mutant ved/biology.organism_classification_rank.species Biology 01 natural sciences Polymerase Chain Reaction Article 03 medical and health sciences CRISPR Animals Multiplex DNA Breaks Double-Stranded Allele Model organism Genotyping Zebrafish Genetics Gene Editing Multidisciplinary ved/biology Cas9 Mice Mutant Strains 030104 developmental biology Exogenous DNA CRISPR-Cas Systems 010606 plant biology & botany |
| Zdroj: | Scientific Reports |
| ISSN: | 2045-2322 |
| Popis: | CRISPR/Cas9 genome-editing has emerged as a powerful tool to create mutant alleles in model organisms. However, the precision with which these mutations are created has introduced a new set of complications for genotyping and colony management. Traditional gene-targeting approaches in many experimental organisms incorporated exogenous DNA and/or allele specific sequence that allow for genotyping strategies based on binary readout of PCR product amplification and size selection. In contrast, alleles created by non-homologous end-joining (NHEJ) repair of double-stranded DNA breaks generated by Cas9 are much less amenable to such strategies. Here we describe a novel genotyping strategy that is cost effective, sequence specific and allows for accurate and efficient multiplexing of small insertion-deletions and single-nucleotide variants characteristic of CRISPR/Cas9 edited alleles. We show that ligation detection reaction (LDR) can be used to generate products that are sequence specific and uniquely detected by product size and/or fluorescent tags. The method works independently of the model organism and will be useful for colony management as mutant alleles differing by a few nucleotides become more prevalent in experimental animal colonies. |
| Databáze: | OpenAIRE |
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