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

1362–1372 Nucleic Acids Research, 2019, Vol. 47, No. 3 doi: 10.1093/nar/gky1216 Published online 5 December 2018 Allele-specific genome editing using CRISPR–Cas9 is associated with loss of heterozygosity in diploid yeast Arthur R. Gorter de Vries , Lucas G.F. Couwenberg, Marcel van den Broek, Pilar de la Torre Cortés, Jolanda ter Horst, Jack T. Pronk and Jean-Marc G. Daran * Department of Biotechnology, Delft University of Technology, Delft 2629HZ, The Netherlands Received August 22, 2018; Revised November 20, 2018; Editorial Decision November 21, 2018; Accepted November 22, 2018 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 antiproliferative and pro-apoptotic genes is a known path to cancer. INTRODUCTION CRISPR–Cas9-assisted genome editing requires the simultaneous presence of the Cas9 endonuclease and a guideRNA (gRNA) that confers target-sequence specificity (1). A gRNA consists of a structural domain and a variable sequence homologous to the targeted sequence (1–4). A Cas9–gRNA complex introduces a DSB when the gRNA binds to its reverse complement sequence on the 5 side of a PAM sequence (NGG). Imperfect gRNA complementarity and/or absence of a PAM sequence strongly reduce editing efficiencies (5). CRISPR–Cas9 enables specific editing of any sequence proximal to a PAM sequence, with minimal off-targeting effects (5). The introduction of a DSB facilitates genome editing by increasing the rate of repair by homologous recombination (6). When a repair fragment consisting of a DNA oligomer with homology to regions on both sides of the introduced DSB is added, it is integrated at the targeted locus by homologous recombination, resulting in replacement of the original sequence and repair of the DSB (2–4). In Saccharomyces cerevisiae, double stranded DNA oligomers with 60 bp of homology are sufficient to obtain accurate gene-editing in almost 100% of transformed cells (3). By inserting sequences between the homologous regions of the repair oligonucleotide, heterozygous sequences of up to 35 kb could be inserted at targeted loci (7). While such gene editing approaches have been very efficient in haploid and homozygous diploid yeasts, the accurate introduction of short DNA fragments can be tedious in heterozygous yeast. In homozygous diploid and polyploid eukaryotes, CRISPR–Cas9 introduces DSBs in all alleles of a targeted sequence (8). In heterozygous genomes, gRNAs can be designed for allele-specific targeting if heterozygous loci have different PAM motifs and/or different 5 sequences close to a PAM motif (8,9), enabling allelespecific gene editing using Cas9. In such cases, a DSB is introduced in only one of the homologous chromosomes while the other homolog remains intact. However, the presence of intact homologous chromosomes facilitates repair of DSBs by homology-directed repair (HDR) using mechanisms such as homologous recombination (HR), or breakinduced repair (BIR) (10–12). In particular, HDR of DSBs can induce chromosome recombinations and even loss of heterozygosity (LOH) in diploid genomes (9,13–16). Therefore, the presence of an intact homologous chromosome could compete with an intended gene-editing event, resulting in reduced editing efficiency and possibly in extensive genetic changes due to LOH. So far, no systematic analysis has been performed of the efficiency of Cas9-mediated * To whom correspondence should be addressed. Tel: +31152782412; Email:  C The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact ABSTRACT Nucleic Acids Research, 2019, Vol. 47, No. 3 1363 gene editing at heterozygous loci. To investigate if Cas9 gene editing works differently in heterozygous diploid yeast, we tested if allele-specific targeting of heterozygous loci using Cas9 enables accurate gene editing in an interspecies Saccharomyces hybrid, and investigated the resulting transformants. In addition, we systematically investigated the efficiency of Cas9-mediated genome editing when targeting various homozygous and heterozygous loci in diploid laboratory S. cerevisiae strains while monitoring genetic changes. Strains, plasmids, primers and statistical analysis S. cerevisiae strains used in this study are derived from the laboratory strains CEN.PK113-7D and S288C (17,18). Yeast strains, plasmids and oligonucleotide primers used in this study are provided in Tables S3–S5. Statistical significance was determined using two-tailed unpaired Student’s ttests in GraphPad Prism 4 (GraphPad, La Jolla, CA, USA). Media and growth conditions Plasmids were propagated overnight in Escherichia coli XL1-Blue cells in 10 ml LB medium containing 10 g/l peptone, 5 g/l Bacto Yeast extract, 5 g/l NaCl and 100 mg/l ampicillin at 37◦ C. Unless indicated otherwise, yeast strains were grown at 30◦ C and 200 RPM in 100 ml flat-bottom flasks containing 50 ml YPD medium, containing 10 g/l Bacto yeast extract, 20 g/l Bacto peptone and 20 g/l glucose. Alternatively, strains were grown in synthetic medium (SM) containing 3.0 g/l KH2 PO4 , 5.0 g/l (NH4 )2 SO4, 0.5 g/l MgSO4 7H2 O, 1 ml/l trace elements, 1 ml/l vitamin solution and 20 g/l glucose (19). For uracil auxotrophic strains, SM-derived media were supplemented with 150 mg/l uracil (20). Solid media were supplemented with 20 g/l agar. Selection for the amdSYM marker was performed on SMAC: SM medium with (...truncated)