Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress
The industrially important non-conventional yeast <i>Komagataella phaffii</i> 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...
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| Format: | Article |
| Language: | English |
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MDPI AG
2022-09-01
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| Series: | Journal of Fungi |
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| Online Access: | https://www.mdpi.com/2309-608X/8/10/992 |
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| author | Veronika Schusterbauer Jasmin E. Fischer Sarah Gangl Lisa Schenzle Claudia Rinnofner Martina Geier Christian Sailer Anton Glieder Gerhard G. Thallinger |
| author_facet | Veronika Schusterbauer Jasmin E. Fischer Sarah Gangl Lisa Schenzle Claudia Rinnofner Martina Geier Christian Sailer Anton Glieder Gerhard G. Thallinger |
| author_sort | Veronika Schusterbauer |
| collection | DOAJ |
| description | The industrially important non-conventional yeast <i>Komagataella phaffii</i> 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 <i>de novo</i> off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites. |
| first_indexed | 2024-03-09T19:59:51Z |
| format | Article |
| id | doaj.art-48be46a237994a869938cf2a1695419d |
| institution | Directory Open Access Journal |
| issn | 2309-608X |
| language | English |
| last_indexed | 2024-03-09T19:59:51Z |
| publishDate | 2022-09-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Journal of Fungi |
| spelling | doaj.art-48be46a237994a869938cf2a1695419d2023-11-24T00:46:20ZengMDPI AGJournal of Fungi2309-608X2022-09-0181099210.3390/jof8100992Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in DistressVeronika Schusterbauer0Jasmin E. Fischer1Sarah Gangl2Lisa Schenzle3Claudia Rinnofner4Martina Geier5Christian Sailer6Anton Glieder7Gerhard G. Thallinger8bisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, AustriaInstitute of Biomedical Informatics, Graz University of Technology, Stremayrgasse 16, 8010 Graz, Austriabisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, AustriaInstitute of Biomedical Informatics, Graz University of Technology, Stremayrgasse 16, 8010 Graz, AustriaThe industrially important non-conventional yeast <i>Komagataella phaffii</i> 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 <i>de novo</i> off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites.https://www.mdpi.com/2309-608X/8/10/992CRISPR/Cas technologynon-conventional yeastsgenome analysisnon-homologous end joining |
| spellingShingle | Veronika Schusterbauer Jasmin E. Fischer Sarah Gangl Lisa Schenzle Claudia Rinnofner Martina Geier Christian Sailer Anton Glieder Gerhard G. Thallinger Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress Journal of Fungi CRISPR/Cas technology non-conventional yeasts genome analysis non-homologous end joining |
| title | Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress |
| title_full | Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress |
| title_fullStr | Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress |
| title_full_unstemmed | Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress |
| title_short | Whole Genome Sequencing Analysis of Effects of CRISPR/Cas9 in <i>Komagataella phaffii</i>: A Budding Yeast in Distress |
| title_sort | whole genome sequencing analysis of effects of crispr cas9 in i komagataella phaffii i a budding yeast in distress |
| topic | CRISPR/Cas technology non-conventional yeasts genome analysis non-homologous end joining |
| url | https://www.mdpi.com/2309-608X/8/10/992 |
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