PT  - JOURNAL ARTICLE
AU  - Cassidy Petree
AU  - Gaurav K. Varshney
TI  - MutliFRAGing: Rapid and Simultaneous Genotyping of Multiple Alleles in a Single Reaction
AID  - 10.1101/523837
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 523837
4099  - http://biorxiv.org/content/early/2019/01/21/523837.short
4100  - http://biorxiv.org/content/early/2019/01/21/523837.full
AB  - The powerful and simple RNA-guided CRISPR/Cas9 technology is a versatile genome editing tool that has revolutionized targeted mutagenesis. CRISPR-based genome editing has enabled large-scale functional genetic studies through the generation of gene knockouts in a variety of model organisms including zebrafish. CRISPR/Cas9 can also be used to target multiple genes simultaneously. One of the challenges associated with applying this technique to zebrafish in a high-throughput manner is the absence of a cost-effective method by which to identify mutants. To address this, we optimized the high-throughput, high-resolution fluorescent PCR-based fragment analysis method to develop MultiFRAGing, a robust and cost-effective method for genotyping of multiple targets in a single reaction. Our approach can identify indels in 4 targets from a single reaction, which represents a four-fold increase in genotyping throughput. This method can be used by any laboratory with access to capillary electrophoresis base sequencing equipment.