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

Abstract

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 βC1 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.

CRISPR/Cas-Mediated Resistance against Viruses in Plants

“…Recently, monopartite begomovirus (cotton leaf curl virus; CLCuV) and associated betasatellite DNA in N. benthamiana plants were targeted for editing [25]. Targeting Rep of CLCuV and βC1 of betasatellite DNA by multiplex approach showed lower virus titers and delay in symptom development when compared with the plants inoculated only with sgRNA targeting Rep.…”

“…This technology has some advantages over artificial microRNAs and RNAi for engineering virus resistance in plants by the disruption of essential viral or host gene instead of silencing those genes at the RNA level. Several studies have demonstrated the advantages of the CRISPR/Cas9 system to effectively confer resistance to DNA and RNA viruses in plants, either by directly targeting and cleaving the viral genome or by modifying the host genes [18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37] (Table 1 and Figure 1). Since the CRISPR-Cas system has a prokaryotic origin, the chances of plant viruses evolving mechanisms to antagonize it are unlikely, in contrast to RNAi, which is a plant-derived process and against which most viruses already carry suppressors [15,38].…”

Application of CRISPR/Cas for Diagnosis and Management of Viral Diseases of Banana

“…Recently, complete resistance against the cotton leaf curl virus (CLCuV, genus Begomovirus ) was demonstrated in N. benthamiana using a CRISPR/Cas9 system by multiplexing gRNAs targeting Rep and IR sequences ( Yin et al, 2019 ). Later, N. benthamiana plants agroinfiltrated with CRISPR/Cas9 construct having multiplex gRNAs targeting the Rep , and β C1 gene of the betasatellites showed delayed disease symptoms and lower titer of CLCuV ( Khan et al, 2020 ). Interestingly, the inactivation of the AC2 gene encoding the transcription activator protein and the AC3 gene encoding the replication enhancer protein of African cassava mosaic virus (ACMV, genus Begomovirus ) failed to produce resistance against the virus in transgenic cassava ( Mehta et al, 2019 ).…”

Next-Generation Sequencing and the CRISPR-Cas Nexus: A Molecular Plant Virology Perspective

“…A broad-spectrum resistance was achieved by targeting the IR region of three distinct begomoviruses, including cotton leaf curl Kokhran virus (CLCuKoV), TYLCV, and merremia mosaic virus (MeMV) ( Ali et al, 2016 ). In another study, the simultaneous targeting of CLCuKoV-encoded Rep and βC1 of cotton leaf curl Multan betasatellite (CLCuMuB) via CRISPR-Cas9 system led to symptom attenuation and manyfold reduction in the viral titer ( Khan et al, 2020 ). The simultaneous targeting of two regions, Rep and IR, of cotton leaf curl Multan virus (CLCuMuV) similarly produced almost complete resistance to CLCuMuV in N. benthamiana plants ( Yin et al, 2019 ).…”