GENOME EDITING BY CRISPR/CAS9 FOR POTYVIRUS RESISTANCE IN TOMATO
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Date
2022-12-28
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University of Agricultural Sciences, Bangalore
Abstract
Genome editing of agriculture crops can be accomplished by using the components of the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9) technology. Here, we present the development of RNAguided Cas9 system for genome editing in tomato cv. ‘Arka Vikas’. We have selected eukaryotic translation initiation factor (eIF) gene family, including eIF4E and its paralogue eIF(iso)4E. Following assembly of sgRNA, ligation should be used to ligate an assembled sgRNA expression cassette into vector. Primers created and the method used to assemble a sgRNA expression cassettes works. A 4140 bp Cas9 forming construct comprising eukaryotic initiation factor 4E genes fragments was mobilized into pBI121 binary vector between CaMV 35S promoter and Nos terminator and designated as Cas9-
ChiVMV binary vector. This construct was mobilized into Agrobacterium tumefaciens (EHA 105 cells) for Agrobacterium-mediated transformation to generate Cas9-ChiVMV transgenic tomato cv. ‘Arka Vikas’ plants. The transgenic plants were screened for the presence of Cas9-ChiVMV by PCR analysis. The CRISPR/Cas9 binary vector targeting the 4E genes was then transformed into tomato plants by Agrobacterium tumefaciensmediated transformation, resulting in efficient target gene editing in the T0 generation. The reporter was activated by Chilli Venial Mottel virus which was engineered to contain sgRNA for the system. However, the results suggest that viral recombination of the sgRNA insert can lead to a loss of the effect over time. A valuable tool for future plant CRISPR/Cas9 development will be the positive readout transactivation system developed in this thesis. We demonstrated the effectiveness of an optimized RNA-guided Cas9 system that can be used for generating homozygous knockout mutants in the progeny of transgenic tomato cv. ‘Arka vikas’ plants