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).