Abstract
Varietal differences within a species with agronomic importance are often based on minor changes in the genomic sequence. For example, fungus-resistant and fungus-susceptible wheat varieties may vary in only one amino acid. The situation is similar with the reporter genes Gfp and Yfp where two base pairs cause a shift in the emission spectrum from green to yellow. Methods of targeted double-strand break induction now allow this exchange precisely with the simultaneous transfer of the desired repair template. However, these changes rarely lead to a selective advantage that can be used in generating such mutant plants. The protocol presented here allows a corresponding allele replacement at the cellular level using ribonucleoprotein complexes in combination with an appropriate repair template. The efficiencies achieved are comparable to other methods with direct DNA transfer or integration of the corresponding building blocks in the host genome. They are in the range of 35 percent, considering one allele in a diploid organism as barley and using Cas9 RNP complexes.
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Acknowledgement
This work was supported by funding from the Federal Ministry of Education and Research (BMBF) under project ID 031B0547. We are grateful to the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben for providing excellent working conditions for our research.
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Becker, M., Hensel, G. (2023). Ribonucleoprotein (RNP)-Mediated Allele Replacement in Barley (Hordeum vulgare L.) Leaves. In: Yang, B., Harwood, W., Que, Q. (eds) Plant Genome Engineering. Methods in Molecular Biology, vol 2653. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3131-7_13
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DOI: https://doi.org/10.1007/978-1-0716-3131-7_13
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