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Phytologist Letters Forum 299 mutants are expected to shed further light on the functional redundancy of the 4CL family. An advantage of gRNA stacking is the simplicity of construct preparation, since a functional Cas9 is already present in existing mutants. The frequent occurrence of SNPs in outcrossing species represents an underappreciated impediment for efficient genome editing. While some studies recommended the use of at least two gRNAs per gene (Ran et al., 2013), we show that one carefully selected gRNA can be effective for study systems with a demanding transformation process. To assess the SNP interference of gRNA specificity, we adopted the CRISPR-P pipeline (Lei et al., 2014) for gRNA design using a custom, variant-substituted 717 genome based on c. 209 resequencing data. Off-target cleavage potential was assessed as described (Hsu et al., 2013), and up to 10 highest-scoring gRNA …