Engineering Resistance Against Cotton Leaf Curl Kokhran Virus-Burewala Strain Using CRISPR-Cas9 System in Nicotiana Benthamiana

Abstract

\n Clustered regularly interspaced palindromic repeats (CRISPR) and associated Cas9 nuclease (CRISPR-Cas9) systems provide adaptive immunity to prokaryotes against infectious phage particles that can be engineered as a genome-editing tool. Guided by an RNA strand, the class II type II CRISPR-Cas9 system can be employed to provide resistance against plant DNA viruses. Here we describe an efficient CRISPR-Cas9 genome editing system based on simultaneous targeting of the highly conserved intergenic region (IR) of the virus that can provide resistance against Cotton leaf curl Kokhran virus-Burewala strain (CLCuKoV-Bur) in Nicotiana benthamiana plants. The data revealed that upon infection, the transgenic plants harboring CRISPR-Cas9 and two gRNAs showed complete resistance against CLCuKoV-Bur/Cotton leaf curl Multan betasatellite (CLCuMB). All efforts failed to find the intact virus in CLCuKoV-Bur/CLCuMB challenged transgenic (OX:Cas9NB:IR) plants using either gene specific PCR primers or CLCuKoV-Bur as a probe in southern blot hybridization. Thus, our results have demonstrated an efficient CRISPR-Cas9 approach to engineer durable resistance against CLCuKoV-Bur in a model system. The implications of these findings are discussed.