Establishing a CRISPR– Cas-like immune system conferring DNA virus resistance in plants

Many archaea and bacteria have adaptive immune systems in which CRISPR RNAs direct Cas endonucleases to cleave foreign nucleic acids on the basis of sequence complementarity. In recent years, CRISPR-Cas-based systems have been developed as a powerful tool for engineering an endogenous genomic locus in various organisms. Geminiviruses are circular single-stranded DNA (ssDNA) viruses that replicate within the nuclei of plant cells, causing serious damage to many dicotyledonous crop plants, including tomato, cassava, cotton, sugar beet and pepper. During geminivirus replication, the ssDNA is converted to a double-stranded DNA (dsDNA) intermediate, from which new ssDNA is generated by rolling-circle replication. In this study, we sought to use sgRNA- Cas9 to specifically target the dsDNA of a geminivirus as a way of inhibiting virus replication and confer-ring virus resistance to host plants. We tested resistance to beet severe curly top virus (BSCTV) in Nicotiana benthamiana and Arabidopsis thaliana. They designed sgRNA specifically targeting to BSCTV genome and found that some sgRNA-Cas9 constructs reduced viral DNA load by up to 97%. This resulted in the systematic relief of the severe leaf-curling symptom. In transgenic plants, the expression of sgRNA-Cas9 significantly reduced the accumulation of BSCTV by destructing the virus genome, and the anti-viral efficiency was correlated with the expression level of Cas9. Our study indicate that this sgRNA-Cas9 immune system has great potential to be used in other host plants to confer resistance to this and other DNA viruses.