Abstract
Clustered, regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR/Cas) based genome editing technology has been developed from the adaptive immune system used by many bacteria and archaea for combating against viruses. Here, we utilize the Cas9 with dual gRNAs designed to target two essential regions of the single-stranded DNA genome of the Cotton Leaf Curl Multan virus (CLCuMuV) which can cleavage the corresponding DNA sequence and thus interrupt viral infection. The transgenic plants expressing Cas9 and dual gRNAs that target different regions of the CLCuMuV genome confer complete resistance to virus infection, thereby demonstrating a novel approach for engineering resistance to geminiviruses.
Highlights
There are several strategies to engineer resistance against viral diseases in plants, the most powerful of which is based on pathogen-derived resistance (PDR), a concept referring to the insertion of viral sequences in plant cells leading to virus resistance (Beachy 1997)
Once the Cas9 and guide RNA (gRNA) co-expressed in the cell, the fsGFP DNA was targeted and cut by the Cas9
The resulting double strand break (DSB) were repaired by non-homologous end joining (NHEJ) and fsGFP could possibly be restored to a functional GFP gene
Summary
There are several strategies to engineer resistance against viral diseases in plants, the most powerful of which is based on pathogen-derived resistance (PDR), a concept referring to the insertion of viral sequences in plant cells leading to virus resistance (Beachy 1997). CRISPR/Cas9-based genome editing technology was recently developed from a defense mechanism used by bacteria and archaea for control of viruses. This system is composed of two components: a programmable DNA nuclease Cas and a short RNA called guide RNA (gRNA). When the complex finds the target, the Cas will cut both strands of the target DNA at around 3 bp upstream of the PAM After this technology was first demonstrated to serve a function in human cells, it was extended and applied to many other species including mouse, worm, fly, fish and plant (Hsu et al 2014)
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