دورية أكاديمية
أعرض في EDS

Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.

التفاصيل البيبلوغرافية
العنوان: Probing gene function in Candida albicans wild-type strains by Cas9-facilitated one-step integration of two dominant selection markers: a systematic analysis of recombination events at the target locus.
المؤلفون: Ramírez-Zavala B; Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany., Hoffmann A; Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany., Krüger I; Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany., Schwanfelder S; Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany., Barker KS; Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA., Rogers PD; Department of Pharmacy and Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA., Morschhäuser J; Institute of Molecular Infection Biology, University of Würzburg, Würzburg, Germany.
المصدر: MSphere [mSphere] 2024 Jul 30; Vol. 9 (7), pp. e0038824. Date of Electronic Publication: 2024 Jun 28.
نوع المنشور: Journal Article
اللغة: English
بيانات الدورية: Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 101674533 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 2379-5042 (Electronic) Linking ISSN: 23795042 NLM ISO Abbreviation: mSphere Subsets: MEDLINE
أسماء مطبوعة: Original Publication: Washington, DC : American Society for Microbiology, [2015]-
مواضيع طبية MeSH: Candida albicans*/genetics , Candida albicans*/drug effects , CRISPR-Cas Systems* , Loss of Heterozygosity* , Recombination, Genetic*, Gene Deletion ; Drug Resistance, Fungal/genetics ; Antifungal Agents/pharmacology ; Fluconazole/pharmacology ; Hygromycin B/pharmacology ; CRISPR-Associated Protein 9/genetics ; Gene Editing/methods ; Streptothricins/pharmacology ; Genetic Markers
مستخلص: The adaptation of gene deletion methods based on the CRISPR-Cas9 system has facilitated the genetic manipulation of the pathogenic yeast Candida albicans , because homozygous mutants of this diploid fungus can now be generated in a single step, allowing the rapid screening of candidate genes for their involvement in a phenotype of interest. However, the Cas9-mediated double-strand breaks at the target site may result in an undesired loss of heterozygosity (LOH) on the affected chromosome and cause phenotypic alterations that are not related to the function of the investigated gene. In our present study, we harnessed Cas9-facilitated gene deletion to probe a set of genes that are constitutively overexpressed in strains containing hyperactive forms of the transcription factor Mrr1 for a possible contribution to the fluconazole resistance of such strains. To this aim, we used gene deletion cassettes containing two different dominant selection markers, caSAT1 and HygB , which confer resistance to nourseothricin and hygromycin, respectively, for simultaneous genomic integration in a single step, hypothesizing that this would minimize undesired LOH events at the target locus. We found that selection for resistance to both nourseothricin and hygromycin strongly increased the proportion of homozygous deletion mutants among the transformants compared with selection on media containing only one of the antibiotics, but it did not avoid undesired LOH events. Our results demonstrate that LOH on the target chromosome is a significant problem when using Cas9 for the generation of C. albicans gene deletion mutants, which demands a thorough examination of recombination events at the target site.
Importance: Candida albicans is one of the medically most important fungi and a model organism to study fungal pathogenicity. Investigating gene function in this diploid yeast has been facilitated by the adaptation of gene deletion methods based on the bacterial CRISPR-Cas9 system, because they enable the generation of homozygous mutants in a single step. We found that, in addition to increasing the efficiency of gene replacement by selection markers, the Cas9-mediated double-strand breaks also result in frequent loss of heterozygosity on the same chromosome, even when two different selection markers were independently integrated into the two alleles of the target gene. Since loss of heterozygosity for other genes can result in phenotypic alterations that are not caused by the absence of the target gene, these findings show that it is important to thoroughly analyze recombination events at the target locus when using Cas9 to generate gene deletion mutants in C. albicans .
Competing Interests: The authors declare no conflict of interest.
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معلومات مُعتمدة: R01 AI058145 United States AI NIAID NIH HHS; R21 AI058145 United States AI NIAID NIH HHS; 210879364 Deutsche Forschungsgemeinschaft (DFG)
فهرسة مساهمة: Keywords: CRISPR-Cas9; Candida albicans; gene deletion; homozygous mutants; loss of heterozygosity
المشرفين على المادة: 0 (Antifungal Agents)
8VZV102JFY (Fluconazole)
3XQ2233B0B (Hygromycin B)
EC 3.1.- (CRISPR-Associated Protein 9)
54003-27-9 (Streptothricins)
0 (Genetic Markers)
تواريخ الأحداث: Date Created: 20240628 Date Completed: 20240730 Latest Revision: 20240801
رمز التحديث: 20240801
مُعرف محوري في PubMed: PMC11288041
DOI: 10.1128/msphere.00388-24
PMID: 38940507
قاعدة البيانات: MEDLINE
الوصف
تدمد:2379-5042
DOI:10.1128/msphere.00388-24