Abstract
In this protocol, we describe the establishment of a CRISPR/Cas9 system in Trichoderma reesei by generating a specific, codon-optimized Cas9-expressing strain and by in vitro transcription of a gRNA. This system induces mutagenesis or introduces a gene in a targeted way based on PEG-mediated protoplast transformation. Up to three targets, multiplexed genome editing can be obtained in one transformation.
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