Most of our knowledge relating to molecular mechanisms of human fungal pathogenesis in Candida albicans relies on reverse genetics approaches, requiring strain engineering. DNA-mediated transformation of C. albicans has been described as highly mutagenic, potentially accentuated by the organism’s genome plasticity, including the acquisition of genomic rearrangements, notably upon exposure to stress. The advent of CRISPR-Cas9 has vastly accelerated the process of genetically modifying strains, especially in diploid (such as C. albicans) and polyploid organisms. The effects of unleashing this nuclease within the genome of C. albicans are unknown, although several studies in other organisms report Cas9-associated toxicity and off-target DNA breaks. Upon the construction of a C. albicans strain collection, we took the opportunity to compare strains which were constructed using CRISPR-Cas9-free and CRISPR-Cas9-dependent transformation strategies, by quantifying and describing transformation-induced loss-of-heterozygosity and hyperploidy events. Our analysis of 57 strains highlights the mutagenic effects of transformation in C. albicans, regardless of the transformation protocol, but also underscores interesting differences in terms of genomic changes between strains obtained using different transformation protocols. Indeed, although strains constructed using the CRISPR-Cas9-free transformation method display numerous concomitant genomic changes randomly distributed throughout their genomes, the use of CRISPR-Cas9 leads to a reduced overall number of genome changes, particularly hyperploidies. Overall, in addition to facilitating strain construction by reducing the number of transformation steps, the CRISPR-Cas9-dependent transformation strategy in C. albicans appears to limit transformation-associated genome changes.

中文翻译：

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使用 CRISPR-Cas9 靶向同源重组可限制白色念珠菌中转化诱导的基因组变化。


我们关于人类白色念珠菌真菌发病机制的分子机制的大部分知识都依赖于反向遗传学方法，需要菌株工程。 DNA介导的白色念珠菌转化被描述为具有高度诱变性，可能因生物体的基因组可塑性而加剧，包括基因组重排的获得，特别是在暴露于压力时。 CRISPR-Cas9的出现极大地加速了菌株基因改造的进程，尤其是二倍体（如白色念珠菌）和多倍体生物。尽管在其他生物体中进行的一些研究报告了 Cas9 相关的毒性和脱靶 DNA 断裂，但在白色念珠菌基因组中释放这种核酸酶的影响尚不清楚。在构建白色念珠菌菌株库后，我们借此机会通过量化和描述转化诱导的杂合性缺失和超倍体来比较使用无 CRISPR-Cas9 和 CRISPR-Cas9 依赖转化策略构建的菌株事件。我们对 57 种菌株的分析强调了白色念珠菌转化的诱变效应，无论转化方案如何，但也强调了使用不同转化方案获得的菌株之间在基因组变化方面的有趣差异。事实上，尽管使用无 CRISPR-Cas9 转化方法构建的菌株显示出许多随机分布在整个基因组中的伴随基因组变化，但 CRISPR-Cas9 的使用导致基因组变化总数减少，特别是超倍体。 总体而言，除了通过减少转化步骤数量来促进菌株构建之外，白色念珠菌中依赖于 CRISPR-Cas9 的转化策略似乎限制了与转化相关的基因组变化。