Abstract
Site-specific genome engineering approaches were greatly facilitated by the recent emergence of the CRISPR-Cas system, enabling precise induction of DNA double-strand breaks. However, up to now its application was mostly restricted to nonhomologous end-joining-mediated targeted mutagenesis. In contrast, precise genome modifications using a suitable donor sequence for homologous still pose a particular challenge in plants, as NHEJ is the dominant repair mechanism for DSBs in somatic cells. To achieve efficient HR-mediated genome modifications in plants, we recently developed the in planta gene targeting (ipGT) system, which works via the induction of DSBs by Cas9 to activate the target and the targeting vector at the same time, making it independent of high transformation efficiencies. Here, we describe an updated protocol of ipGT for the model plant Arabidopsis, taking into account our recent improvements based on egg-cell-specific expression of Staphylococcus aureus Cas9.
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Abbreviations
- CRISPR-Cas:
-
Clustered regularly interspaced short palindromic repeats-CRISPR associated
- DSB:
-
Double-strand break
- GT:
-
Gene targeting
- ipGT:
-
In planta GT
- NHEJ:
-
Nonhomologous end joining
- PPT:
-
Phosphinothricin
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Wolter, F., Huang, TK., Puchta, H. (2020). Efficient Homologous Recombination-Mediated in Planta Gene Targeting by Egg-Cell-Specific Expression of Staphylococcus aureus Cas9 from Arabidopsis. In: Islam, M.T., Bhowmik, P.K., Molla, K.A. (eds) CRISPR-Cas Methods . Springer Protocols Handbooks. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0616-2_2
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DOI: https://doi.org/10.1007/978-1-0716-0616-2_2
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