Whole genome sequencing analysis of effects of CRISPR/CAS9 in Komagataella phaffii

CRISPR/Cas is an effective tool for genome engineering. Especially in the industrially important non-conventional yeast Komagataella phaffii, CRISPR offers a low effort and more efficient alternative to methods relying mainly on homologous recombination. We conducted whole genome sequencing on 147 CRISPR/Cas9 engineered strains derived from 3 different K. phaffii platform strains, where the introduced double strand breaks were sealed by cellular NHEJ repair. We included single gene target and multiplexing experiments on two genes. Furthermore, we targeted different stretches of a large region which only contained genes likely to be non-essential. The detailed analysis of repair events showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target sites, especially in multiplexing experiments. The multiplexing of the putatively non-essential region led to a truncation of chromosome 3 in multiple cases. The targeted deleted region contained more than 100 kbp of genomic sequence, which coded for 20 genes and several repeats of ribosomal DNA. Off-target mutations were rare and randomly distributed, although a considerable number appeared within gene coding regions. This indicates that off-target mutations are connected rather to random mutagenesis than to unspecific binding of the guide RNA.