CRISPR/Cas9 Tool Kit for Efficient and Targeted Insertion/Deletion Mutagenesis of the Komagataella phaffii (Pichia pastoris) Genome.

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.