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

Abstract

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...

Homozygous might be hemizygous: CRISPR/Cas9 editing in iPSCs results in detrimental on-target defects that escape standard quality controls

“…In the most comprehensive study to date, Weisheit et al (2020) examined 35 iPSC clones edited across 3 genomic locations and identified on-target defects in 18%-40% of cases. Additionally, CRISPR/Cas9 can cause chromosomal instability, which can result in copy loss or copy-neutral LOH (Alateeq et al, 2018;Gorter de Vries et al, 2019;Hajiahmadi et al, 2019;Korablev et al, 2020;Ledford, 2020;Prat et al, 2020;Przewrocka et al, 2020;Rayner et al, 2019;Weisheit et al, 2020;Yang et al, 2017;Zischewski et al, 2017). Importantly, genotyping using PCR and Sanger sequencing of a short DNA fragment serves well as an initial screen, but may lead to false identification of edited clones, as large on-target insertions/deletions may cause exclusive amplification of only one allele.…”