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CRISPR/Cas9 is a powerful tool for genome engineering. However, so far it could not be applied to engineer the chloroplast genome, because chloroplasts lack the machinery for non-homologous end joining. As the consequence, double-strand DNA breaks are lethal in chloroplasts. This problem has been recently overcome by developing the prime editor (PE) tool that requires nicking only one of the two DNA strands. The PE is a mutant Cas9 fused with a reverse transcriptase domain. The efficiency of repair is ensured by fusing the guide RNA with the repair template (pegRNA) that is provided for reverse transcriptase. The project will involve construction of the PE and pegRNAs and testing them by induction of point mutations in the chloroplast genome that confer antibiotic and herbicide resistance. The tools will be useful to improve photosynthetic efficiency and increasing tolerance to adverse conditions.
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