Watson Rebekah G, Hole Camaron R
Department of Clinical Pharmacy and Translational Science, The University of Tennessee Health Science Center, Memphis, Tennessee, USA.
mSphere. 2025 Apr 29;10(4):e0107024. doi: 10.1128/msphere.01070-24. Epub 2025 Apr 2.
infections are a significant cause of morbidity and mortality among AIDS patients and the third most common invasive fungal infection in organ transplant recipients. The cryptococcal cell wall is very dynamic and can be modulated depending on growth conditions. It was reported that when is grown in unbuffered yeast nitrogen base (YNB) for 48 hours, the pH of the media drastically drops, and the cells start to shed their cell walls. With this observation, we sought to determine if YNB-grown cells could be used directly for genetic transformation. To test this, we targeted using TRACE (transient CRISPR-Cas9 coupled with electroporation) in YNB-grown or competent cells. Deletion of the gene results in red-pigmented colonies, allowing visual confirmation of disruption. We were able to successfully delete in YNB-grown cells with better efficiency compared to competent cells. Recent studies have shown that gene deletion can be accomplished using short (50 bp) homology arms in place of the normal long arms (~1 kb). However, it was inefficient, leading to more insertions and gene disruption than gene deletions. We tested short homology with YNB-grown cells vs. competent cells and found that gene deletion was significantly improved in YNB-grown cells, at around 60% compared to 6% in competent cells. This was also observed when we deleted with the short arms. Altogether, using simple growth conditions, we have greatly improved the speed and efficiency of cryptococcal genetic transformations.IMPORTANCEThe World Health Organization recently ranked as the highest-priority fungal pathogen based on unmet research and development needs and its public health importance. Understanding cryptococcal pathogenicity is key for developing treatments. We found that using simple growth conditions can greatly improve the speed and efficiency of cryptococcal genetic transformations. This finding will advance the field by expanding the ease of cryptococcal genetic manipulations.
感染是艾滋病患者发病和死亡的重要原因，也是器官移植受者中第三常见的侵袭性真菌感染。隐球菌细胞壁非常动态，可根据生长条件进行调节。据报道，当在无缓冲的酵母氮源（YNB）中培养48小时时，培养基的pH值会急剧下降，细胞开始脱落细胞壁。基于这一观察结果，我们试图确定在YNB中培养的细胞是否可直接用于基因转化。为了验证这一点，我们在YNB中培养的细胞或感受态细胞中使用TRACE（瞬时CRISPR-Cas9与电穿孔相结合）靶向[具体基因]。[具体基因]的缺失会导致红色色素菌落的出现，从而可以通过视觉确认基因的破坏。与感受态细胞相比，我们能够在YNB中培养的细胞中更高效地成功缺失[具体基因]。最近的研究表明，使用短（50 bp）同源臂代替正常的长同源臂（约1 kb）也可以完成基因缺失。然而，这种方法效率低下，导致插入和基因破坏的情况比基因缺失更多。我们测试了YNB中培养的细胞与感受态细胞使用短同源臂的情况，发现YNB中培养的细胞的基因缺失情况有显著改善，约为60%，而感受态细胞中为6%。当我们使用短臂缺失[另一个具体基因]时也观察到了这种情况。总之，通过简单的生长条件，我们极大地提高了隐球菌基因转化的速度和效率。
世界卫生组织最近根据未满足的研发需求及其公共卫生重要性，将[隐球菌]列为最优先的真菌病原体。了解隐球菌的致病性是开发治疗方法的关键。我们发现，使用简单的生长条件可以极大地提高隐球菌基因转化的速度和效率。这一发现将通过提高隐球菌基因操作的便利性推动该领域的发展。
