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.

Fundamental questions regarding the minimal requirements for life have prompted scientists to embark on top-down efforts to reduce microbial genomes to the minimum set of genes and proteins necessary to sustain cell survival and division. While these efforts are generally focused on small, prokaryotic genomes, Saccharomyces cerevisiae , a popular industrial and model organism, has a typical eukaryotic genome characterized by a high genetic redundancy.

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterised by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of minor paralogs and complicates modular and combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, pentose phosphate pathway, tricarboxylic acid cycle and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 from the 111 genes yielded a minimal CCM strain, which despite the elimination of 32 % of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes of the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodelling of yeast CCM.

7 Targeted DNA double-strand breaks (DSBs) with CRISPR-Cas9 have revolutionized genetic 8 modification by enabling efficient genome editing in a broad range of eukaryotic systems. Accurate 9 gene editing is possible with near-perfect efficiency in haploid or (predominantly) homozygous 10 genomes. However, genomes exhibiting polyploidy and/or high degrees of heterozygosity are less 11 amenable to genetic modification. Here, we report an up to 99-fold lower gene editing efficiency when 12 editing individual heterozygous loci in the yeast genome. Moreover, Cas9-mediated introduction of a 13 DSB resulted in large scale loss of heterozygosity affecting DNA regions up to 360 kb that resulted in 14 introduction of nearly 1700 off-target mutations, due to replacement of sequences on the targeted 15 chromosome by corresponding sequences from its non-targeted homolog. The observed patterns of 16 loss of heterozygosity were consistent with homology directed repair. The extent and frequency of 17 loss of heterozygosity represent a novel mutagenic side-effect of Cas9-mediated genome editing, 18 which would have to be taken into account in eukaryotic gene editing. In addition to contributing to the 19 limited genetic amenability of heterozygous yeasts, Cas9-mediated loss of heterozygosity could be 20 particularly deleterious for human gene therapy, as loss of heterozygous functional copies of anti-21 proliferative and pro-apoptotic genes is a known path to cancer.22 30 DSB facilitates genome editing by increasing the rate of repair by homologous recombination (6).31 When a repair fragment consisting of a DNA oligomer with homology to regions on both sides of the 32 introduced DSB is added, it is integrated at the targeted locus by homologous recombination, 33 resulting in replacement of the original sequence and repair of the DSB (2-4). In S. cerevisiae, double 34 stranded DNA oligomers with 60 bp of homology are sufficient to obtain accurate gene-editing in 35 almost 100% of transformed cells (3). By inserting sequences between the homologous regions of the 36 1 repair oligonucleotide, heterozygous sequences of up to 35 Kbp could be inserted at targeted loci (7). 37While such gene editing approaches have been very efficient in haploid and homozygous diploid 38 yeasts, the accurate introduction of short DNA fragments can be tedious in heterozygous yeast. In 39 homozygous diploid and polyploid eukaryotes, CRISPR-Cas9 introduces DSBs in all alleles of a 40 targeted sequence (8). In heterozygous genomes, gRNAs can be designed for allele-specific targeting 41 if heterozygous loci have different PAM motifs and/or different 5' sequences close to a PAM motif 42 (8,9), enabling allele-specific gene editing using Cas9. In such cases, a DSB is introduced in only one 43 of the homologous chromosomes while the other homolog remains intact. However, the presence of 44 intact homologous chromosomes facilitates repair of DSBs by homologous recombination (HR), 45 homology-directed repair (HDR) or break-induced repair (BIR) in eukaryotes (10-1...