Engineering Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen

Abstract

Artificial microRNA (amiRNA) technology offers reversible and flexible gene inactivation and complements genome-editing technologies. However, obtaining transgenic plants with maximal gene silencing remains a major technical challenge in current amiRNA applications. Here, we incorporated an empirically determined feature of effective amiRNAs to the amiRNA design and in silico generated a database containing 533,429 gene-specific amiRNAs for silencing 27,136 genes in Arabidopsis (Arabidopsis thaliana), with a genome coverage of 98.87%. In both single-gene and multiple-gene silencing, we observed an overall improvement in performance by amiRNAs designed using our strategy in Arabidopsis protoplasts and transgenic plants. In addition, the endogenous tRNA-processing system was used to generate multiple amiRNAs from tRNA-pre-amiRNA tandem repeats for multiplex gene silencing. An intronic amiRNA-producing fluorescent reporter was explored as a visual screening strategy for transgenic Arabidopsis and rice (Oryza sativa) plants with maximal whole-plant or cell type-specific gene silencing. These improvements enable the amiRNA technology to be a functional gene knockout tool for basic and applied plant research.

MicroRNAs and new biotechnological tools for its modulation and improving stress tolerance in plants

“…The main drawbacks of this strategy are the selection of backbone or pre‐amiRNA sequences for effective silencing without any off‐target effects (Carbonell et al ., ). For major target specificity, mature amiRNAs must have low sequence similarity with nontarget genes (Zhang et al ., ).…”

“…However, these approaches produce a diverse set of siRNAs that might potentially silence nontarget genes (resulting in off‐target effects). To overcome this problem, an artificial MIR gene (amiRNA) strategy was developed to produce specific miRNAs and effectively silence target genes (Zhang et al ., ). These amiRNAs have a conserved secondary foldback structure similar to that of a typical pre‐miRNA.…”

“…Constitutive or transient expression of (a) artificial MIR genes to accumulate artificial mi RNA (ami RNA ) and the depletion of the target mRNA (Zhang et al ., ). In brief, the ami RNA gene under the control of a typical promoter is transcribed by RNA polymerase II in the nucleus, and primary transcripts are similarly processed to canonical mi RNA s, originating primary ami RNA (pri‐ami RNA ).…”

Plant miRNA Cross-Kingdom Transfer Targeting Parasitic and Mutualistic Organisms as a Tool to Advance Modern Agriculture

“…The knowledge acquired on endogenous miRNAs as regulators of gene functions within and among organisms led researchers to develop increasingly sophisticated agricultural technologies based on miRNAs. Among these, the amiRNA (artificial miRNA) strategy was developed to produce specific miRNAs that can effectively silence designated genes (Zhang et al, 2018). One of the main characteristics of amiRNAs is the conserved secondary foldback structure that has to be similar to that of a typical pre-miRNA.…”

The working dead: repurposing inactive CRISPR-associated nucleases as programmable transcriptional regulators in plants

“…To date, the CRISPR/Cas system is still limited for cell/tissue-specific or conditional manipulation of gene activity (Zhang et al 2018a). It is readily conceivable that both dCas-TRDs (e.g., dCas-3 9 SRDX) and dCas-TADs (e.g., dCas9-TV) can be expressed under a cell/ tissue-specific or chemically inducible promoter to confer spatial or temporal control of gene repression and activation, which can perfectly complement the CRISPR/Cas system for flexible gene manipulation in plants.…”