Check CRISPR Editing Events in Transgenic Wheat with Next-Generation Sequencing

Abstract

CRISPR-Cas9 is a convenient tool to create knockdown mutants for desired genes. Early generations of transgenic plants usually have multiple editing events. To select the best plants for future study, these plants need to be screened for editing type and proportion. However, the traditional restriction enzyme method cannot tell the editing type and Sanger sequencing cannot check a mixture of different editing events. Next-generation sequencing (NGS) is the best choice for screening CRISPR-Cas9 induced edits. With NGS, hundreds to thousands of T1 plants can be sequenced without genome-specific primers. Here we present the detailed procedure for using NGS to select plants with the best editing, including primer design, PCR setup, sample preparation, and sequencing result analysis.