Conditional knockdown of gene expression in plants via 3' UTR editing.

The spatiotemporal knockdown of genes through genome editing heralds a new frontier in molecular breeding, yet it remains largely unexplored. Recognizing the intricate regulatory networks of endogenous microRNAs (miRNAs), we posited that integration of specific miRNA target sequences into the 3' untranslated region (UTR) of a gene could construct artificial miRNA-dependent regulatory circuits, facilitating precise spatiotemporal gene suppression. To test this hypothesis, we selected three endogenous miRNAs with unique expression profiles by analyzing rice miRNA expression profiles. Results from both transient assays and stably-edited rice plants confirmed that in-locus incorporation of miRNA targets into the 3' UTR of target genes can substantially reduce their expression in a spatiotemporal manner. Using GID1 as a target gene, we found that knockin of the miR156a target led to a remarkable 97% constitutive reduction; knockin of the tissue-specifically expressed miR396c target significantly reduced its expression in shoots alone; and knock-in of the long-day-induced miR528 target triggered a dramatic and temporal decrease of 95% specifically under such light exposure. These findings underscore the viability of miRNA-mediated, in-locus knockdown (MiRKD) as a convenient approach for crop breeding, leveraging miRNA expression traits and genome editing for conditional gene suppression.