Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

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, ge...

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Bibliographic Details
Published inbioRxiv
Main Authors Arthur R Gorter De Vries, Couwenberg, Lucas G F, Van Den Broek, Marcel, Pilar De La Torre Cortes, Jolanda Ter Horst, Pronk, Jack T, Daran, Jean-Marc G
Format Paper
LanguageEnglish
Published Cold Spring Harbor Cold Spring Harbor Laboratory Press 22.08.2018
Subjects
Apoptosis
CRISPR
Deoxyribonucleic acid
DNA
DNA damage
Double-strand break repair
Gene therapy
Genomes
Heterozygosity
Homology
Loss of heterozygosity
Mutation
Polyploidy
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Summary: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 that resulted in introduction of nearly 1700 off-target mutations, 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.
DOI:10.1101/397984