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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.
We developed a novel parallel quantitative PCR (qPCR) assay for reliable genotyping of genome editing outcomes like CRISPR-Cas, ZFNs, and TALENs. This method simplifies the detection of mutations, including insertions or deletions (indels).
Area of Science:
- Molecular Biology
- Genetics
- Biotechnology
Background:
- CRISPR-Cas, ZFNs, and TALENs enable precise gene editing for functional studies.
- Detecting point mutations and indels from genome editing is challenging with conventional PCR.
- Existing genotyping methods can be complex and not easily scalable.
Purpose of the Study:
- To develop a simple and reliable method for genotyping genome editing outcomes.
- To create a parallel qPCR assay for efficient detection of mutations.
- To enable large-scale and automated genotyping analysis.
Main Methods:
- Developed a parallel qPCR assay incorporating an iGenotype index.
- Utilized allele-specific primers for consistent iGenotype values (1, 0, -1).
- Employed an R program for the analysis of qPCR data.
Main Results:
- The iGenotype index provided constant values across allele-specific primers.
- The assay demonstrated simple and reliable genotyping capabilities.
- The R program facilitated large-scale and automated genotyping.
Conclusions:
- The parallel qPCR assay with iGenotype index offers a robust solution for genotyping genome editing.
- This method simplifies the detection of various mutations, including indels.
- The protocol supports efficient, large-scale, and automated genotyping for genetic research.
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