Targeting Non-Coding RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting

Abstract

Non-coding RNAs (ncRNAs) have emerged as versatile master regulator of biological functions in recent years. MicroRNAs (miRNAs) are small endogenous ncRNAs of 18-24 nucleotides in length that originates from long self-complementary precursors. Besides their direct involvement in developmental processes, plant miRNAs play key roles in gene regulatory networks and varied biological processes. Alternatively, long ncRNAs (lncRNAs) are a large and diverse class of transcribed ncRNAs whose length exceed that of 200 nucleotides. Plant lncRNAs are transcribed by different RNA polymerases, showing diverse structural features. Plant lncRNAs also are important regulators of gene expression in diverse biological processes. There has been a breakthrough in the technology of genome editing, the CRISPR-Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) technology, in the last decade. CRISPR loci are transcribed into ncRNA and eventually form a functional complex with Cas9 and further guide the complex to cleave complementary invading DNA. The CRISPR-Cas technology has been successfully applied in model plants such as Arabidopsis and tobacco and important crops like wheat, maize, and rice. However, all these studies are focused on protein coding genes. Information about targeting non-coding genes is scarce. Hitherto, the CRISPR-Cas technology has been exclusively used in vertebrate systems to engineer miRNA/lncRNAs, but it is still relatively unexplored in plants. While briefing miRNAs, lncRNAs and applications of the CRISPR-Cas technology in human and animals, this review essentially elaborates several strategies to overcome the challenges of applying the CRISPR-Cas technology in editing ncRNAs in plants and the future perspective of this field.

Keywords: CRISPR-Cas; NHEJ; homologous recombination; lncRNA; miRNA; off-target mutation.

FIGURE 1

Classification and major functions of various non-coding RNAs.

FIGURE 2

Series of events to generate a clustered regulatory interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) mutagenised plant. CRISPR-Cas mediated genome engineering in plants requires ‘single guide’ RNA (sgRNA) and CRISPR-associated protein 9 (Cas9). Specially designed sgRNA are expressed under a small RNA promoter and transfected along with a Cas9 expression plasmid, to form a complex which targets complementary DNA adjacent to the protospacer adjacent motif (PAM). sgRNA:Cas9 complex generates a double strand break (DSB) that may either be repaired precisely (without any effect) or imperfectly leading to a mutation (indel) in the genomic target sequence.