CRISPR/CAS9 Mediated Knock out of Multiple Trichome-Regulating MYB Transcription Factors in Populus

The Populus tremula x P. alba (IRNA 717-1B4) PtaMYB186 transcription factor was previously identified as a regulator of trichome initiation during early stages of leaf development. Here, we employ CRISPR/Cas9 to target PtaMYB186 and its close paralogs, PtaMYB138 and PtaMYB38, for mutagenesis in Populus tremula x P. alba using a single gRNA targeting a conserved region. We obtained 37 transgenic events, all with a glabrous phenotype. Amplicon-sequencing confirmed successful edits in all 25 randomly selected mutants, with 24 complete knockouts. RNA-Seq analysis of fully expanded mutant leaves identified 244 and 308 genes that were significantly up- and down-regulated, respectively, when compared with controls. Interestingly, two orthologs of Arabidopsis trichome-regulating genes were up-regulated and four orthologs of Arabidopsis circadian rhythm genes were down-regulated. The data suggest that mutation of trichome regulators normally expressed during early stages of leaf development can have pleiotropic effects on the transcriptomes of more mature leaves. Gas chromatography–mass spectrometry analysis of leaf cuticle wax composition revealed no major compositional differences, but subtle changes were observed between mutant and control plants. Five mutants were subjected to whole genome re-sequencing and preliminary assessments revealed no evidence of off-target mutations or rearrangements by CRISPR/Cas9 in poplar. Finally, the recently completed genome sequencing of Populus tremula x P. alba revealed copy number variation that may be linked to natural variation in trichome density among Populus species. This research demonstrates that a single variant-free gRNA is highly efficient for multi-allele knockout in poplar. The striking glabrous phenotype of the mutants also highlights the potential of targeting trichome-regulators as a non-lethal visual marker system for assessing new CRISPR reagents or mutagenesis strategies.