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 integrating specific miRNA target sequences into the 3' 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 stable edited rice plants confirmed that in-locus incorporation of miRNA targets into the 3' UTR of target genes substantially reduced their expression in a spatiotemporal manner. Specifically, taking GID1 as the target gene, we found that knock-in of miR156a target led to a remarkable 97% constitutive reduction
knock-in of tissue-specifically expressed miR396c target significantly diminished its expression in shoots alone
and knock-in of the long-day induced miR528 target triggered a dramatic and temporal decrease by 95% specifically under such light exposure. These findings underscore the viability of this miRNA-mediated in-locus knockdown (MiRKD) as a convenient approach for crop breeding, leveraging miRNA expressional traits and genome editing for conditional gene suppression.