A Genotyping Protocol to Identify CRISPR/Cas9-Edited Events in Hexaploid Wheat

Abstract

Targeted genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become an established method to introduce changes into a plant genome. While traditional genetic modification allows us to follow transgenic events with selection markers as they are often co-integrated, this is not always feasible when Cas9/guide RNA-based methods are applied. In cases where genome editing does not result in a selectable phenotype, the only option to analyze editing events is by sequencing the target region through either conventional or high-throughput sequencing, both of which can be tedious and costly. In this chapter, we present a rapid, economic genotyping method that was designed to identify editing events in the three wheat sub-genomes. The method is not only economical but is in general very useful when dealing with polyploid organisms where identification and characterization of edited events can be challenging.