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Protocol for streamlining genotyping of germline-transmissible mutants from genome editing by using a parallel
Emeric M Louis1, Liezhen Fu2, Nga Luu2
1Section on Molecular Morphogenesis, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA; Unité Mixte de Recherche 7221, Département Adaptation du Vivant, Centre National de la Recherche Scientifique, Muséum National d'Histoire Naturelle, Alliance Sorbonne Universités, Paris, France.
Abstract:
Targeted genome editing using CRISPR-Cas, ZFNs, or TALENs enables precise gene function studies but often produces point mutations or insertions or deletions (indels) that are difficult to detect by conventional PCR. We developed a parallel qPCR assay with an iGenotype index for simple, reliable genotyping. iGenotype values (1, 0, -1) remained constant across allele-specific primers. qPCR data can be analyzed via an R program, enabling large-scale or automated genotyping. For complete details on the use and execution of this protocol, please refer to Fu et al.1.
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