CRISPR-Activation: Boosting Expression of Plant MIRs

In the field of plant genome editing, the CRISPR/Cas system stands out as an exceptionally versatile tool. Originally conceived for gene editing, the catalytically inactive Cas9 variant, known as dead Cas9 (dCas9), takes on a new role when coupled with transcriptional effectors, offering a finely tuned mechanism for controlling gene expression. This innovation has paved the way for the evolution of sophisticated systems such as CRISPR-Act 2.0 and CRISPR-Act 3.0, which significantly enhance transcriptional activation in plants. This chapter serves as an introduction to a protocol that harnesses CRISPR-activation (CRISPRa) to modulate the transcription of plant miRNA genes. Recognizing the imperative for meticulous construct design and promoter identification, the protocol outlines steps encompassing vector construction. It underscores the critical importance of comprehending miRNA promoter regions and cis-acting elements, presenting a holistic approach for effectively employing CRISPRa in manipulating miRNA expression to modulate traits in plants.