Dataset Information

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

ABSTRACT: Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb and up to 1700 heterozygous nucleotides, due to replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of loss of heterozygosity were consistent with homology directed repair. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-proliferative and pro-apoptotic genes is a known path to cancer.

SUBMITTER: Gorter de Vries AR

PROVIDER: S-EPMC6379674 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

Gorter de Vries Arthur R AR Couwenberg Lucas G F LGF van den Broek Marcel M de la Torre Cortés Pilar P Ter Horst Jolanda J Pronk Jack T JT Daran Jean-Marc G JG

Nucleic acids research 20190201 3

Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when editing individual hete ...[more]

PMID: 30517747

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Project description:Genetic manipulation is often used to study gene function. However, unplanned genome changes (including single nucleotide polymorphisms [SNPs], aneuploidy, and loss of heterozygosity [LOH]) can affect the phenotypic traits of the engineered strains. Here, we compared the effect of classical deletion methods (replacing target alleles with selectable markers by homologous recombination) with CRISPR-Cas9 editing in the diploid human-pathogenic yeast Candida parapsilosis. We sequenced the genomes of 9 isolates that were modified using classic recombination methods and 12 that were edited using CRISPR-Cas9. As a control, the genomes of eight isolates that were transformed with a Cas9-expressing plasmid in the absence of a guide RNA were also sequenced. Following gene manipulation using classic homologous recombination, only one strain exhibited extensive LOH near the targeted gene (8.9 kb), whereas another contained multiple LOH events not associated with the intended modification. In contrast, large regions of LOH (up to >1,100 kb) were observed in most CRISPR-Cas9-edited strains. LOH most commonly occurred adjacent to the Cas9 cut site and extended to the telomere in four isolates. In two isolates, we observed LOH on chromosomes that were not targeted by CRISPR-Cas9. Among the CRISPR-edited isolates, two exhibited cysteine and methionine auxotrophy caused by LOH at a heterozygous site in MET10, approximately 11 and 157 kb downstream from the Cas9 target site, respectively. C. parapsilosis isolates have relatively low levels of heterozygosity. However, our results show that mutation complementation to confirm observed phenotypes is required when using CRISPR-Cas9. IMPORTANCE 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. We show that the Cas9-induced double-strand break is often associated with loss of heterozygosity in the asexual diploid human fungal pathogen Candida parapsilosis. This can result in deleterious heterozygous variants (e.g., stop gain in one allele) becoming homozygous, resulting in unplanned phenotypic changes. Our results stress the importance of mutation complementation even when using CRISPR-Cas9.

Dataset Information

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

Publications

Allele-specific genome editing using CRISPR-Cas9 is associated with loss of heterozygosity in diploid yeast.

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