Abstract
The CRISPR/Cas9 system has recently been used to confer molecular immunity against several eukaryotic viruses, including plant DNA geminiviruses. Here, we provide a detailed analysis of the efficiencies of targeting different coding and non-coding sequences in the genomes of multiple geminiviruses. Moreover, we analyze the ability of geminiviruses to evade the CRISPR/Cas9 machinery. Our results demonstrate that the CRISPR/Cas9 machinery can efficiently target coding and non-coding sequences and interfere with various geminiviruses. Furthermore, targeting the coding sequences of different geminiviruses resulted in the generation of viral variants capable of replication and systemic movement. By contrast, targeting the noncoding intergenic region sequences of geminiviruses resulted in interference, but with inefficient recovery of mutated viral variants, which thus limited the generation of variants capable of replication and movement. Taken together, our results indicate that targeting noncoding, intergenic sequences provides viral interference activity and significantly limits the generation of viral variants capable of replication and systemic infection, which is essential for developing durable resistance strategies for long-term virus control.
Highlights
Geminiviruses threaten food security and agriculture, infecting key crop species, especially in tropical and subtropical regions[1]
Our results indicate that Clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9)-induced variants in open reading frames (ORFs) of geminiviruses were capable of replication and systemic movement, thereby evading the CRISPR/Cas[9] machinery, but the intergenic regions (IRs) variants failed to replicate and move systemically
To deliver single guide RNA (sgRNA) specific to the coding regions of the genes encoding the coat protein (CP) and the divalent cation coordination RCRII domain of replication associated protein (Rep) (Fig. 1A) of Cotton Leaf Curl Kokhran Virus (CLCuKoV), the sgRNAs were delivered through Tobacco rattle virus (TRV) RNA2 into Cas9OE Nicotiana benthamiana plants[30]
Summary
Geminiviruses threaten food security and agriculture, infecting key crop species, especially in tropical and subtropical regions[1]. Recent work showed that site-specific nucleases can directly target and cleave the viral genome. This cleavage of the viral genome leads to the generation of double strand breaks (DSBs), which are either repaired by the imprecise non-homologous end-joining repair (NHEJ) machinery or by precise homology-directed repair (HDR)[6,7,8]. We demonstrated that the CRISPR/Cas[9] machinery can target coding and non-coding sequences of different geminiviruses[7,27,28]. This targeting results in reduced viral accumulation and delayed or abolished symptoms. We investigated the replicative ability of mutated viral variants that could evade the CRISPR/Cas[9] system
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