Abstract
The CRISPR/Cas9-based system for targeted mutagenesis has become an indispensable tool for functional genetics in plants. CRISPR/Cas9 allows users to generate loss-of-function alleles in genes of interest with precision and in a simple-to-use system. This manuscript outlines important points to consider for experimental design and utilization of CRISPR/Cas9 in targeted mutagenesis in maize. It also introduces the pRGEB32-BAR vector modified for use in maize that allows simultaneous delivery of multiple gRNAs using a simple assembly. Vector selection, gRNA design, genetic strategies, and genotyping approaches are discussed, with an emphasis on achieving isolation of homozygous mutant plants in a time- and cost-efficient manner.