Manipulation of miRNAs for breeding insect and disease-free crops
Knock-out mutation of miRNAs provides an opportunity to validate their target genes. Traditional methods of mutagenesis (chemical/radiations/insertional) are largely ineffective due to the small size of miRNAs. Additionally, miRNAs belong to the conserved family comprising multiple members with a potentially redundant structure and function. Therefore, loss-of-function analysis of miRNA genes has become a major challenge. Currently, target mimics (TMs), short tandem target mimics (STTMs), molecular sponges (SPs), and artificial miRNAs (amiRNAs) are the most commonly used techniques for loss-of-function analysis of miRNAs. With the availability of genome editing technology, precise modification of particular loci in the genome has become possible (Voytas 2013). Site-specific nuclease (SSN) of the genome editing tools induces double-stranded breaks (DSBs) at a predefined site of the locus. DSBs are subsequently repaired by the endogenous DNA repair system of the plant. DSBs are repaired by two major mechanisms, non-homologous end-joining (NHEJ) and homology-directed repair (HDR). NHEJ predominantly occurs in plants that are relatively error-prone, leading to small insertions or deletions (InDels) at the target locus (Lieber 2010; Symington & Gautier 2011; Puchta 2017). Alternatively, HDR is more precise than NHEJ but less frequent, involving a donor template containing homologous regions matching the target locus.
In recent years, Clustered Regularly Interspaced Short Palindromic Repeats and associated protein 9 (CRISPR-Cas9) (Jinek et al.2012; Feng et al. 2013; Zhang, Wen & Guo 2014) and CRISPER from Prevotella and Francisella 1 (CRISPR-Cpf1) (Tang et al. 2017) (Tang et al., 2017) have emerged as powerful tools for precise genome engineering. Almost any sequence in the genome can be targeted using specifically designed single ­guide RNAs (sgRNAs). DNA repair via NHEJ facilitates loss-of-function due to the introduction of small random insertions or deletions at the target site. Over the past few years, CRISPR-Cas9 has been widely used for targeted modification of many plant genomes (Shen et al. 2014; Belhaj, Chaparro-Garcia, Kamoun, Patron & Nekrasov 2015; Yin, Gao & Qiu 2017). Previously, post-transcriptional technologies that impound or degrade miRNAs have been used in plants for loss-of-function analysis. However, these methods lead to variable inhibition in transgenic plants (Reichel, Li, Li & Millar 2015).
The feasibility to manipulate the miRNA genes using the CRISPR/Cas9 system has been demonstrated in soybean for miR1514 andmiR1509 (Jacobs, LaFayette, Schmitz & Parrott 2015) and in Arabidopsis for miR169a and miR827 (Zhao et al.2016). Recently, CRISPR/Cas9 was efficiently used for introducing heritable mutations in mature miRNAs of rice (Zhou et al. 2017, 2019a; Bi et al. 2020). Knockout of Osa-miR396e  and Osa-miR396f using the CRISPR/Cas9 system enhanced both grain size and panicle branching under the nitrogen-deficient condition in rice (Zhang et al. 2020a). CRISPR/Cas9-mediated mutagenesis of Osa-miR396ef promotes GA signaling that enhances grain yield by increasing grain size and modulates leaf blade and sheath in rice (Miao, Wang, He, Liu & Zhu 2020). Targeted mutagenesis of single miRNA gene (e.g., Osa-miR408 and Osa-miR528 ) and miRNA gene family (e.g., Osa-miR815 and Osa-miR820 ) has been demonstrated using CRISPR/Cas9 system in rice. Larger deletion in Osa-miR528elevated transcript level of the target genes under salt stress (Zhouet al. 2017). Targeted mutagenesis of OsSPL9 using CRISPR)/Cas9 showed a significant reduction in Osa-miR528expression suggested a critical role of OsSPL9 in transcriptional regulation of Osa-miR528 (Yang et al. 2019).
A series of CRISPR/Cas9-mediated mutations in Osa-miR156 were produced to investigate seed dormancy (Miao et al. 2019). CRISPR/Cas9 system was employed to introduce mutations in the multiplesuperoxide dismutase (SOD) genes to demonstrateOsa-miR398 -mediated resistance to M. oryzae (Li et al. 2019d). Targeted mutations were introduced in the target gene (OsARF12 ) of Osa-miR167d using the CRISPR/Cas9 method to investigate Osa-miR167-ARF12 interaction in immunity against rice blast disease (Zhao et al. 2019).