Abstract
Efficient targeted genome engineering of Komagataella phaffii requires balanced expression of Cas9 nuclease and a target-specific guide RNA (gRNA). In addition, correct processing of the transcribed RNA to provide the designed gRNA as a target selective partner of targeted Cas9 protein for binding to genomic DNA is essential for efficient genome engineering. This method describes a step-by-step procedure and recommended tools for simple and efficient design of gRNAs to introduce insertions or deletions at targeted sites by CRISPR/Cas9-directed double-strand breaks, followed by error-prone nonhomologous end-joining repair.
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Fischer, J.E., Glieder, A. (2022). CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome. In: Mapelli, V., Bettiga, M. (eds) Yeast Metabolic Engineering. Methods in Molecular Biology, vol 2513. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2399-2_8
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DOI: https://doi.org/10.1007/978-1-0716-2399-2_8
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