Sugarcane transgenics expressing MYB transcription factors show improved glucose release

Additional file 1: Figure S1. Nucleotide and amino acid alignments of MYB31 and MYB42. Alignments of published (Fornalé et al. 2006) and cloned nucleotide and amino acid sequences (ORF and UTR) for ZmMYB31 (NM_001112479) and ZmMYB42 (NM_001112539). Alignments were made using the Kyoto University Bioinformatics Center website ( http://www.genome.jp/tools-bin/clustalw ) and amino acid translations were made using the ORF region of each MYB nucleotide sequences and Vector NTI software. The start and stop codons are underlined in nucleotide sequence alignments with the 5′ and 3′ UTR regions being upstream and downstream of the start and stop codons respectively. The R2 and R3 motifs in each sequence are underlined with light gray and dark grey shading respectively

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar