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.
