Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA.
Highlights
Plant viruses invade their host plants to replicate and propagate, severely affecting plant growth and yield, causing significant losses in crop quality and quantity, and seriously threatening food security around the world [1,2,3,4]
We investigated the ability of Clustered regularly interspaced short palindromic repeats (CRISPR)/pCas13a to provide durable and heritable interference against the Turnip mosaic virus (TuMV)-GFP virus in Arabidopsis thaliana plants
We previously reported the successful utilization of CRISPR/LshCas13a to target and interfere with the TuMV-GFP RNA virus in N. benthamiana plants [26]
Summary
Plant viruses invade their host plants to replicate and propagate, severely affecting plant growth and yield, causing significant losses in crop quality and quantity, and seriously threatening food security around the world [1,2,3,4] Combatting such devastating losses requires efforts to develop novel technologies to control plant viruses. Different strategies to engineer virus resistance have been developed over the years, including transgenic expression of viral and non-viral factors [5,6,7,8,9]. These technologies are promising, many hurdles have complicated their utility in agriculture [10,11]. Class II CRISPR/Cas systems are the most widely developed and used systems for RNA and DNA targeting and regulation, owing to their dependence on two simple components, a Cas effector protein that mediates cleavage of the target RNA or DNA, and a guide RNA (gRNA) that guides the Cas
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