Extensive loss of heterozygosity upon CRISPR-Cas9 mediated genome editing in yeast. Saccharomyces cerevisiae strain:IMX1585 | breed:IMX1555 X S288C

RNA-guided introduction of double-strand breaks (DSBs) in DNA by CRISPR-Cas9 has revolutionized genetic modification by enabling efficient genome editing in many eukaryotic systems including fungi, plants and mammalian cells. Accurate gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous genomes. However, genomes that exhibit polyploidy and/or high degrees of heterozygosity are less amenable to genetic modification. Here, we report up to 1697 off-target mutations and an up to 99-fold lower efficiency of accurate gene-editing when editing individual heterozygous loci in the yeast genome. Introduction of DSBs at heterozygous loci resulted in replacement of sequences on the targeted chromosome by corresponding sequences from its non-targeted homologous chromosome. These replacements ranged in size from single nucleotides to entire chromosome arms. The observed patterns of loss of heterozygosity were consistent with homology-directed repair, which is highly conserved among eukaryotes and responsible for allele propagation using gene drives and allele-specific gene editing. The extent and frequency of loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, which needs to be taken into account in eukaryotic gene editing. 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