title: Detection of nucleotide-specific CRISPR/Cas9 modified alleles using multiplex ligation detection.
abstract: 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.
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: R
authors.initials: R
authors.lastname: Kc
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: A
authors.initials: A
authors.lastname: Srivastava
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: J M
authors.initials: JM
authors.lastname: Wilkowski
authors.affiliation: Department of Pediatrics and Communicable Diseases, Division of Pediatric Hematology/Oncology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: C E
authors.initials: CE
authors.lastname: Richter
authors.affiliation: Department of Pediatrics and Communicable Diseases, Division of Pediatric Hematology/Oncology, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: J A
authors.initials: JA
authors.lastname: Shavit
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: D T
authors.initials: DT
authors.lastname: Burke
authors.affiliation: Department of Human Genetics, University of Michigan Medical School, Ann Arbor, Michigan, USA.
authors.forename: S L
authors.initials: SL
authors.lastname: Bielas
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D053903:DNA Breaks, Double-Stranded; D059766:DNA End-Joining Repair; D000072669:Gene Editing / Q000379:methods; D060005:Genotyping Techniques / Q000379:methods*; D008817:Mice, Mutant Strains; D016133:Polymerase Chain Reaction / Q000379:methods*; D015027:Zebrafish / Q000235:genetics