CRISPR/Cas9‐Based Multiplex Genome Editing in Monocot and Dicot Plants

Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9‐mediated genome targeting system has been applied to a variety of organisms, including plants. Compared to other genome‐targeting technologies such as zinc‐finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs), the CRISPR/Cas9 system is easier to use and has much higher editing efficiency. In addition, multiple “single guide RNAs” (sgRNAs) with different target sequences can be designed to direct the Cas9 protein to multiple genomic sites for simultaneous multiplex editing. Here, we present a procedure for highly efficient multiplex genome targeting in monocot and dicot plants using a versatile and robust CRISPR/Cas9 vector system, emphasizing the construction of binary constructs with multiple sgRNA expression cassettes in one round of cloning using Golden Gate ligation. We also describe the genotyping of targeted mutations in transgenic plants by direct Sanger sequencing followed by decoding of superimposed sequencing chromatograms containing biallelic or heterozygous mutations using the Web‐based tool DSDecode. © 2016 by John Wiley & Sons, Inc.