Abstract
BackgroundGeminiviruses cause damaging diseases in several important crop species. However, limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. Recently, the bacterial CRISPR/Cas9 system has been transferred to plants to target and confer immunity to geminiviruses. In this study, we use CRISPR-Cas9 interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses.ResultsOur results show that the CRISPR system fails to confer effective resistance to the virus during glasshouse inoculations. Further, we find that between 33 and 48% of edited virus genomes evolve a conserved single-nucleotide mutation that confers resistance to CRISPR-Cas9 cleavage. We also find that in the model plant Nicotiana benthamiana the replication of the novel, mutant virus is dependent on the presence of the wild-type virus.ConclusionsOur study highlights the risks associated with CRISPR-Cas9 virus immunity in eukaryotes given that the mutagenic nature of the system generates viral escapes in a short time period. Our in-depth analysis of virus populations also represents a template for future studies analyzing virus escape from anti-viral CRISPR transgenics. This is especially important for informing regulation of such actively mutagenic applications of CRISPR-Cas9 technology in agriculture.
Highlights
Geminiviruses cause damaging diseases in several important crop species
CRISPR-Cas9 transgenic plants failed to demonstrate effective geminivirus resistance, and we found that the use of CRISPR-Cas9 led to emergence of a novel, conserved mutant virus that cannot be cleaved by CRISPR-Cas9
We chose sgRNA1, which targets both the viral AC2 gene coding for the multifunctional TrAP protein involved in gene activation, virus pathogenicity, and suppression of gene silencing, and the AC3 gene coding for the REn protein involved in replication enhancement [13] (Fig. 1a, b)
Summary
Geminiviruses cause damaging diseases in several important crop species. limited progress has been made in developing crop varieties resistant to these highly diverse DNA viruses. We use CRISPR-Cas interference in the staple food crop cassava with the aim of engineering resistance to African cassava mosaic virus, a member of a widespread and important family (Geminiviridae) of plant-pathogenic DNA viruses. The bacterial CRISPR-Cas (clustered, regularly interspaced short palindromic repeats-CRISPR associated 9) gene editing system can be used to engineer resistance to DNA viruses through direct cleavage of the virus genome. Biotechnology has proven effective for engineering cassava mosaic virus resistance by using plant endogenous RNA interference (RNAi) pathways to limit the expression of virus genes [9]. We applied CRISPR-Cas to engineer resistance to geminiviruses, the African cassava mosaic virus (ACMV) (Begomovirus; Geminiviridae) in cassava and investigated the impact of engineering resistance on geminivirus evolution. We urge caution in the application of CRISPR-Cas for virus resistance in plants, both in glasshouse and field settings, to avoid inducing the evolution of resistant viruses
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