Accelerating plant breeding to cope with changing growing conditions requires a high demand for precise and efficient genome editing. Prime editing (PE) is a gene editing method based on a CRISPR–Cas system that allows the assembly of multiple types of precise DNA alterations in vivo with low off-target rates. PE systems have been successfully applied to monocots, however, at the plant level as a whole, the PE performance of dicots is very low and is not actually successful. Improvements have been made to the technology, such as the configuration of PE2max, the protection of "flip and elongate" primer editing guide RNA (pegRNA), the reverse transcriptase (RT) variant truncated by RNase H, the nucleocapsid (NC) RNA chaperone assistance, the design of primer binding sites in the optimal melting temperature range, the use of pairs of pegRNAs and complex promoters to drive the transcription of pegRNAs, and more. Despite these advances, the application of PE systems in plants, especially tomatoes, has been challenging due to the self-complementarity of primer binding sites and spacer sequences, the complexity of RT templates (RTTs), the stability of PE proteins, various unfavorable PE reaction conditions, and the stability of pegRNAs.
Recently, Tien Van Vu et al. from the Department of Applied Life Sciences, Plant Molecular Biology and Biotechnology Research Center, Gyeongsang University, Korea, published a research paper entitled "Optimized dicot prime editing enables heritable desired edits in tomato and Arabidopsis" in Nature Plants. Combining the favorable conditions of PE complex loading and PE reaction, the researchers successfully overcame the obstacles to the application of PE system in dicots by using altered engineered pegRNA (epegRNA) variants, new combinations of PE protein components, overproduction of pegRNA by U6 complex promoter, amplification of PE system by twin viral replic, and heat treatment, and improved PE system improved the editing and genetic inheritance of tomato and Arabidopsis. This study fills the gap in the application of PE in plants.
The results showed that the new combination of PE protein components enhanced expectations, the PE tool had a strong detection effect on multiple loci of tomato, the heat treatment significantly improved the activity of PE, the PE tool had a strong detection effect on multiple loci of tomato, and the enhanced PE tool did not increase off-target activity, the paired pegRNAs ensured the highest PE efficiency of tomato, the RNA chaperone further improved the PE performance, and a variety of methods were used to optimize the formation of a new PE tool. More than 600 times more efficient than expected editing.
The study also demonstrated that the ideal PE edit should be stable and genetically stable and exhibit a strong phenotype in terms of herbicide tolerance. This study made significant improvements to PE tools for bifamily, improving editing efficiency and accuracy, and obtaining transgenic-free plants and stable genetic editing types. These advances hold promise for the future of plant gene editing, potentially enabling more precise and efficient modifications, even in species where traditional editing tools are less effective. In addition, further research is needed to validate the effectiveness of these tools in other plant species, including monocots, and to investigate their applicability in a variety of agricultural and horticultural settings.
Original link:
https://www.nature.com/articles/s41477-024-01786-w
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