Generation of Gene Knock-Out Mutants in Ustilago maydis Using Cas9hf Nuclease

Gene disruption of nonessential genes became more convenient with the adaptation of the CRISPR-Cas9 system for Ustilago maydis by the group of Regine Kahmann (MPI Marburg, Germany) in 2016. In our group, we have developed the system further to create defined marker-free gene deletions. Therefore, we used the CRISPR-Cas9 system together with oligonucleotides composed of 40 nucleotides of upstream and downstream flanking regions. Here, we describe the entire way from the decision, which gene of interest should be deleted, to a marker-free U. maydis mutant strain.