RNA Viral Vectors for Accelerating Plant Synthetic Biology

Abstract

The tools of synthetic biology have enormous potential to help us uncover the fundamental mechanisms controlling development and metabolism in plants. However, their effective utilization typically requires transgenesis, which is plagued by long timescales and high costs. In this review we explore how transgenesis can be minimized by delivering foreign genetic material to plants with systemically mobile and persistent vectors based on RNA viruses. We examine the progress that has been made thus far and highlight the hurdles that need to be overcome and some potential strategies to do so. We conclude with a discussion of biocontainment mechanisms to ensure these vectors can be used safely as well as how these vectors might expand the accessibility of plant synthetic biology techniques. RNA vectors stand poised to revolutionize plant synthetic biology by making genetic manipulation of plants cheaper and easier to deploy, as well as by accelerating experimental timescales from years to weeks.

Proteome expansion in thePotyviridaeevolutionary radiation

“…Virus infectious clones can be engineered and optimized as expression vectors for plant biotechnology and synthetic biology (Fig. 12 ) (Pasin, Menzel and Daròs 2019 , Khakhar and Voytas 2021 ). Vectors based on potyvirids have been applied for disparate uses, ranging from production of heterologous peptides in plants, to flowering induction, gene silencing, metabolic engineering, CRISPR/Cas-targeted plant genome editing, and reprogramming of crops and their organelles (Lin et al .…”

CRISPR–Cas-mediated transcriptional control and epi-mutagenesis

“…Transient methods such as agroinfiltration, viral-based delivery approaches, lipid-based systems, or nanomaterial-based techniques have been used for the transient delivery of genome editing systems into plants ( Sandhya et al, 2020 ). So far, only agroinfiltration and viral delivery approaches have been adopted to transiently deliver CRISPR components for regulating gene expression or for epimutagenesis in whole plants ( Piatek et al, 2015 ; Ghoshal et al, 2020 ; Khakhar and Voytas, 2021 ). Delivery of dCas-based reagents by PEG-mediated transformation for transcriptional activation has only been achieved in protoplasts ( Li et al, 2017 ; Lowder et al, 2018 ).…”

“…The first is ensuring the efficient delivery of the CRISPR reagents to the appropriate targeted cells, such as meristematic cells, that facilitate the inheritance of the introduced change ( Altpeter et al, 2016 ). Recent studies have used plant mobile RNA signal sequences that enhance the movement of the RNA viruses to facilitate the delivery of CRISPR reagents into meristematic cells ( Ghoshal et al, 2020 ; Ma et al, 2020 ; Zhang et al, 2020 ; Khakhar and Voytas, 2021 ). The second challenge is the limited cargo size of viral vectors.…”

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana

“…Typically, the yield of the recombinant protein is about 1% of the TSP [32,33]. In whole plants, the problem of low yield can be solved by transient expression or expression of the target gene in chloroplasts [1,8]. To increase the biosynthesis of recombinant proteins in cell suspension culture, researchers use various methods, including optimizing the expression of target genes due to regulatory elements in expression cassettes, reducing the degradation of target proteins, optimizing the conditions for cultivating plant cells, etc.…”

“…In whole plants, the problem of low yield of the target protein can be overcome by transient expression. Transient expression systems have surpassed the challenge of rapid and high-yield expression of recombinant proteins in plants, allowing for gram-sized quantities to be attained in as little as 5 days [7,8]. However, so far only a few examples of the use of a transient expression in suspension cell cultures are known in the literature [9,10].…”