Evaluation of the CRISPR/Cas9 system for the development of resistance against Cotton leaf curl virus in model plants

Over the last decade, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) CRISPR/Cas9 system has been used by biologists in various fields. In plant biology, the technology is being utilised to manufacture transgenic plants resistant to different diseases. In Pakistan, the Cotton leaf curl virus (CLCuV) (a begomovirus) affects cotton plants causing significant loss to the economy of this agriculturally based country. In the present study, we use the CRISPR/Cas9 system in Nicotiana benthamiana Domin (a model plant) to develop resistance against CLCuV. An interesting facet of the study was the comparison of two constructs (pHSE401 and pKSE401) with regards to their efficacy in the virus inhibition. The pKSE401 vector contained a Cas9 nuclease and two guide RNAs (gRNAs), one targeting the Replication associated protein (Rep) gene and the other targeted the beta C1 gene of the Betasatellite. The vector pHSE401 had only one sgRNA that targeted the (Rep) gene. Both genes that are intended to be targeted play important roles in the replication of CLCuV. Plants infiltrated with pKSE401 exhibited a delay in the development of the symptoms of the disease and showed lower virus titres. Our proposed multiplexing approach gave efficient results of the resistance in the model plants, and the results in this communication may be extended to the CRISPR/Cas9 based editing of cotton plants.