CRISPR-Cas9 Editing Induces Loss of Heterozygosity in the Pathogenic Yeast Candida parapsilosis

Lombardi, Lisa; Bergin, Sean A; Ryan, Adam P.; Zuniga-Soto, Evelyn; Butler, Geraldine

doi:10.1128/msphere.00393-22

Cited by 4 publications

(2 citation statements)

References 55 publications

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“…gambiae, despite its importance for public health and the successful development of several gene drive strategies in the lab. CRISPR-Cas9 mediated DSBs have been seen to induce GCTs of up to 1.7kb in yeasts (27,28). I-Sce1 mediated DSBs have induced GCTs of up to 0.5kb in mammalian stem cells (29), and up to 0.8kb (average 471bp) in Drosophila melanogaster (30,31).…”

Section: Dna Repair Mechanisms and Gene Drive Homing In Mosquitoesmentioning

confidence: 99%

Homing-based gene drives can introgress rapidly into local genetic backgrounds with minimal chromosomal conversion in Anopheles gambiae

Nolan,

Pescod,

Bevivino

et al. 2023

Preprint

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CRISPR-Cas9 gene drive control strategies use a homing selfish genetic element which induces a double-stranded break at the target site and is copied into the opposing chromosome, breaking an essential gene and ensuring super-Mendelian inheritance. During the copying process SNPs could potentially be transferred from one chromosome to another, converting tracts of the target to match the recipient. This would reduce the ability of gene drives to escape their genetic background of origin, with potential impacts on carrier fitness and the success of the strategy. In this study we use Anopheles gambiae strains with variation around the target site to determine the extent of chromosomal conversion during gene drive activity and the mechanism of inheritance bias. We confirm a homing-based mechanism and show that two thirds of homing events are resolved within 50 bp, showing that gene drive introgression into a wild strain can occur within one generation.

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Section: Dna Repair Mechanisms and Gene Drive Homing In Mosquitoesmentioning

confidence: 99%

Homing-based gene drives can introgress rapidly into local genetic backgrounds with minimal chromosomal conversion in Anopheles gambiae

Nolan,

Pescod,

Bevivino

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…This can result in loss of heterozygosity (LOH) for extended regions on this chromosome and, consequently, effects on the phenotype of the mutants that are not caused by the deletion of the target gene but by loss of allelic variants of other genes, especially when only one allele is functional ( 4 , 30 – 48 ). In fact, it has recently been shown that CRISPR-Cas9-based gene editing strongly increased the frequency of LOH in Candida parapsilosis , especially on the target chromosome ( 49 ).…”

Section: Introductionmentioning

confidence: 99%

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

Ramírez-Zavala,

Hoffmann,

Krüger

et al. 2024

mSphere

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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 .

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Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts

et al. 2023

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CRISPR-Cas9 Editing Induces Loss of Heterozygosity in the Pathogenic Yeast Candida parapsilosis

Abstract: CRISPR-Cas9 has greatly streamlined gene editing and is now the gold standard and first choice for genetic engineering. However, we show that in diploid species, extra care should be taken in confirming the cause of any phenotypic changes observed.

Cited by 4 publications

References 55 publications

Homing-based gene drives can introgress rapidly into local genetic backgrounds with minimal chromosomal conversion in Anopheles gambiae

Homing-based gene drives can introgress rapidly into local genetic backgrounds with minimal chromosomal conversion in Anopheles gambiae

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

Improving CRISPR-Cas9 mediated genome integration in interspecific hybrid yeasts

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