Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in Komagataella phaffii: A Budding Yeast in Distress

Veronika Schusterbauer, Jasmin E. Fischer, Sarah Gangl, Lisa Schenzle, Claudia Rinnofner, Martina Geier, Christian Sailer, Anton Glieder, Gerhard G. Thallinger*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


The industrially important non-conventional yeast Komagataella phaffii suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified de novo off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites
Original languageEnglish
Article number992
JournalJournal of Fungi
Issue number10
Publication statusPublished - Oct 2022


  • CRISPR/Cas technology
  • genome analysis
  • non-conventional yeasts
  • non-homologous end joining

ASJC Scopus subject areas

  • Microbiology (medical)
  • Ecology, Evolution, Behavior and Systematics
  • Plant Science

Fields of Expertise

  • Human- & Biotechnology
  • Information, Communication & Computing


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