Asian Research Thesis Index

Abstract

During the last decade, progress in genome engineering had a giant leap with huge applications in basic and synthetic biology. The resulting genome engineering revolution, now known as gene editing, was mostly driven by the introduction of engineered endonucleases, specifically zinc finger nucleases (ZFNs), clustered regularly interspaced short palindromic repeats (CRISPR) RNA-guided nucleases (e.g., Cas9) and transcription activator like effector nucleases (TALENs). In higher eukaryotic systems, targeted mutagenesis is frequently achieved by non-homologous end joining (NHEJ) based repair of DNA double-strand breaks (DSB) induced site specifically by engineered nucleases resulting knockdown or malfunction of the targeted genes. Genimiviruses have become a serious threat to a number of crops in Pakistan. This study was initiated with the major objective of demonstrating TALEN technology for suppression of cotton leaf curl virus (CLCuV), a major menace to cotton crop in Pakistan. DNA sequences of five most prevalent strains of cotton leaf curl virus were aligned to identify consensus regions for TALEs/TALEN targeting. TALEs/TALENs were constructed using golden gate cloning strategy. Activity of TALEs/TALENs for virus suppression was successfully demonstrated in Nicotiana benthamiana by challenging with infectious clones of, specifically, cotton leaf curl kokhran virus (CLCuKV). DSBs in the targeted region were determined by T7E1 assay. Virus accumulation was assessed by qPCR and TALEN expression was analyzed by RT-PCR. Inoculated plants showed varying degree of resistance to CLCuKV in three ways; attenuated virus infection, delayed symptoms and lower virus accumulation. Thesis results successfully demonstrate the potential of TALEs/TALEN technology for CLCuV suppression which can be a broader genome editing platform for suppression of other viruses.

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