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Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format
Published on: April 8, 2017
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A simple and practical workflow for genotyping of CRISPR-Cas9-based knockout phenotypes using multiplexed amplicon
Midori Iida1, Miyuki Suzuki2, Yuto Sakane2
1Department of Bioscience and Bioinformatics, Kyushu Institute of Technology, Iizuka, Japan.
Genes to Cells : Devoted to Molecular & Cellular Mechanisms
|April 24, 2020
Summary
This study presents a streamlined genotyping workflow for CRISPR-Cas9 knockout founders using pooled amplicon sequencing and the CLiCKAR web tool. This method enables efficient, large-scale in vivo functional screening of target genes by accurately evaluating somatic mutations.
Area of Science:
- Genetics
- Molecular Biology
- Bioinformatics
Background:
- CRISPR-Cas9 technology facilitates in vivo functional screening of genes.
- Genotyping pooled samples with next-generation sequencing is crucial for large-scale screening.
- Efficient evaluation of Cas9-induced somatic mutations is necessary.
Purpose of the Study:
- To present a simple workflow for genotyping multiple CRISPR-Cas9 knockout founders.
- To enable rapid and scalable functional screening of target genes.
- To introduce the CLiCKAR web tool for data analysis.
Main Methods:
- Pooled amplicon sequencing using custom barcoded primers.
- Illumina MiSeq platform for single-indexed library sequencing.
- The CLiCKAR web tool for demultiplexing and mutation evaluation.
Main Results:
- Successful genotyping of multiple CRISPR-Cas9 knockout founders.
- Efficient demultiplexing of pooled sequence data.
- Practical reports on insertion/deletion positions, frameshift ratio, and mutation counts.
Conclusions:
- The presented workflow simplifies genotyping for CRISPR-Cas9 loss-of-function screening.
- This approach facilitates genotype-phenotype correlation studies in various organisms.
- CLiCKAR enhances the efficiency of analyzing somatic mutations in functional screening.
