Handelman Mariana, Osherov Nir
Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv, Tel-Aviv 69978, Israel.
J Fungi (Basel). 2023 Jun 2;9(6):644. doi: 10.3390/jof9060644.
is a common human fungal pathogen that can cause a range of diseases. Triazoles are used to treat infections, but resistance is increasing due to mutations in genes such as , and overexpression of efflux pumps. Verifying the importance of these mutations is time-consuming, and although the use of CRISPR-Cas9 methods has shortened this process, it still relies on the construction of repair templates containing a selectable marker. Here, employing in vitro-assembled CRISPR-Cas9 along with a recyclable selectable marker, we devised a quick and easy way to effectively and seamlessly introduce mutations conferring triazole resistance in . We used it to introduce, alone and in combination, triazole resistance-conferring mutations in , and . With the potential to seamlessly introduce genes imparting resistance to additional existing and novel antifungals, toxic metals, and environmental stressors, this technique can considerably improve the ability to introduce dominant mutations in .
是一种常见的人类真菌病原体，可引发一系列疾病。三唑类药物用于治疗感染，但由于诸如、等基因的突变以及外排泵的过度表达，耐药性正在增加。验证这些突变的重要性耗时费力，尽管使用CRISPR-Cas9方法缩短了这一过程，但它仍然依赖于构建含有选择标记的修复模板。在此，我们利用体外组装的CRISPR-Cas9以及可循环利用的选择标记，设计了一种快速简便的方法，可有效且无缝地在中引入赋予三唑耐药性的突变。我们用它单独或联合在、和中引入赋予三唑耐药性的突变。由于具有无缝引入赋予对其他现有和新型抗真菌药物、有毒金属及环境应激源耐药性的基因的潜力，该技术可显著提高在中引入显性突变的能力。
