Schusterbauer Veronika, Fischer Jasmin E, Gangl Sarah, Schenzle Lisa, Rinnofner Claudia, Geier Martina, Sailer Christian, Glieder Anton, Thallinger Gerhard G
bisy GmbH, Wuenschendorf 292, 8200 Hofstaetten, Austria.
Institute of Biomedical Imaging, Graz University of Technology, Stremayrgasse 16, 8010 Graz, Austria.
J Fungi (Basel). 2022 Sep 21;8(10):992. doi: 10.3390/jof8100992.
The industrially important non-conventional yeast suffers from low rates of homologous recombination, making site specific genetic engineering tedious. Therefore, genome editing using CRISPR/Cas represents a simple and efficient alternative. To characterize on- and off-target mutations caused by CRISPR/Cas9 followed by non-homologous end joining repair, we chose a diverse set of CRISPR/Cas targets and conducted whole genome sequencing on 146 CRISPR/Cas9 engineered single colonies. We compared the outcomes of single target CRISPR transformations to double target experiments. Furthermore, we examined the extent of possible large deletions by targeting a large genomic region, which is likely to be non-essential. The analysis of on-target mutations showed an unexpectedly high number of large deletions and chromosomal rearrangements at the CRISPR target loci. We also observed an increase of on-target structural variants in double target experiments as compared to single target experiments. Targeting of two loci within a putatively non-essential region led to a truncation of chromosome 3 at the target locus in multiple cases, causing the deletion of 20 genes and several ribosomal DNA repeats. The identified off-target mutations were rare and randomly distributed, with no apparent connection to unspecific CRISPR/Cas9 off-target binding sites.
这种具有重要工业价值的非常规酵母存在同源重组率低的问题，使得位点特异性基因工程操作繁琐。因此，使用CRISPR/Cas进行基因组编辑是一种简单而有效的替代方法。为了表征由CRISPR/Cas9引发并随后通过非同源末端连接修复导致的靶向和脱靶突变，我们选择了一系列不同的CRISPR/Cas靶点，并对146个经CRISPR/Cas9工程改造的单菌落进行了全基因组测序。我们将单靶点CRISPR转化的结果与双靶点实验的结果进行了比较。此外，我们通过靶向一个可能非必需的大基因组区域来研究可能的大缺失程度。对靶向突变的分析显示，在CRISPR靶点位点出现了数量意外之多的大缺失和染色体重排。我们还观察到，与单靶点实验相比，双靶点实验中靶向结构变异有所增加。在一个假定非必需区域内靶向两个位点，在多个案例中导致3号染色体在靶点位点处截断，造成20个基因和几个核糖体DNA重复序列的缺失。所鉴定出的脱靶突变很少且随机分布，与非特异性CRISPR/Cas9脱靶结合位点没有明显关联。
