格特隐球菌（Cryptococcus gattii）属于担子菌酵母，主要存在于土壤、树木和树洞中。它是一种致病真菌，能够感染免疫功能正常且健康的个体。随着格特隐球菌在全球的传播，目前世界上六个大洲均有格特隐球菌感染病例的报道，但是国际上对于它的姊妹种新生隐球菌的研究比较多，对格特隐球菌的研究相对较少。此外，格特隐球菌的分子生物学研究工具匮乏，严重阻碍了对其致病机理的探究。由细菌和古生菌进化而来的获得性免疫系统——CRISPR-Cas9 ，已被设计成为多种生物的基因编辑工具，但其在格特隐球菌中的应用情况尚未报道。本论文率先构建了适用于格特隐球菌的“自杀型”CRISPR-Cas9系统，并将该系统应用于格特隐球菌耐药性的研究中，为后续格特隐球菌基因功能和致病机理的深入探究提供了强有力的技术支撑。

CRISPR-Cas9 系统由具有引导功能的guide RNA 和具有核酸酶活性的 Cas9 两个元件组成。以GenBank 数据库中格特隐球菌的基因组信息为基础，我们首先通过NCBI Blast工具对格特隐球菌U6 snRNA基因进行查找，并选取U6 snRNA的启动子来激活guide RNA表达。以本实验室在新生隐球菌中建立的pRH003 CRISPR-Cas9 质粒为骨架，我们通过表达框替换的方式，利用格特隐球菌肌动蛋白基因ACT1的启动子激活Cas9蛋白的表达，利用翻译延长因子TEF1基因的启动子激活筛选标记基因hph表达。通过酶切验证和DNA测序，我们最终获得了适用于格特隐球菌的pBN003 CRISPR质粒。构建完成后，我们选用报告基因ADE2验证该CRISPR-Cas9系统在格特隐球菌中的有效性。通过在pBN003质粒中引入Cas9作用靶点和目的基因上下游同源臂，我们成功获得了ade2Δ突变体，实现了ADE2基因的重组敲除。同时，通过引入定点突变donor DNA的方式，我们实现了对ADE2基因的定点编辑。综合以上实验结果，我们成功构建了能够高效介导格特隐球菌基因组编辑的pBN003 CRISPR-Cas9系统。

前期研究结果表明，与VGI菌株相比，格特隐球菌中VGII菌株对唑类药物具有更强的耐药性。结合转录组测序分析，我们发现VGII菌株中ABC转运蛋白表达量显著高于VGI菌株。其中，ATM1 和 PXA2 基因是调节格特隐球菌唑类药物敏感的候选靶点。因此，为了验证Atm1和Pxa2 转运蛋白在格特隐球菌耐药性中的潜在生物学作用，我们利用本论文构建的CRISPR-Cas9敲除系统，对ATM1 和 PXA2 基因的生物学功能进行分析。表型检测结果表明，ATM1基因的缺失导致了细胞内铁硫簇合成受到影响，在含有铁离子的培养基上出现生长缺陷，从而降低了细胞对氧化压力的耐受性。atm1Δ菌株在含有醋酸钠的培养基中出现生长缺陷。此外，该突变株在含有0.01%SDS、1%刚果红（Congo Red）以及高渗透压的 YPD培养基上细胞生长能力减弱，此结果说明ATM1基因在细胞壁的合成和稳定中发挥重要作用。由于atm1Δ菌株在高温条件下适应能力减弱以及荚膜直径变小，我们利用隐球菌常用动物模型大蜡螟对突变株进行毒力测定。实验结果表明，侵染atm1Δ菌株的大蜡螟的致死率显著低于野生型，揭示了Atm1在格特隐球菌毒力中的重要作用。在对临床治疗隐球菌病常用抗真菌药物的敏感性进行检测时，我们发现ATM1缺失菌株在含有酮康唑和伊曲康唑的培养基中表现出明显的生长缺陷，尤其是在5-FC存在的条件下，atm1Δ几乎完全无法生长，该结果证实了ATM1基因高表达对菌株耐药性的促进作用，即Atm1转运蛋白参与细胞对抗真菌药物的外排。我们将5-FC处理的ATM1突变株与野生型进行转录组测序，结果表明表达量差异显著的基因主要集中在细胞壁结构、膜的组成、血红素结合、铁硫簇结合这六条通路中，这与我们表型检测实验结果也是一致的，但ATM1缺失导致突变株对5-FC敏感性显著增强的机制尚在研究中。除此之外，通过PCR和Southern blot实验验证，我们也成功获取了PXA2 基因的敲除菌株。但目前的检测条件下，我们尚未发现pxa2Δ明显的表型变化，推测Pxa2蛋白在特定条件下发挥功能。

综上所述，本论文构建了适用于格特隐球菌的CRISPR-Cas9基因编辑系统，该系统为格特隐球菌的基因功能和致病机理研究提供了强有力的技术保障。此外，我们还应用该系统研究了基因 ATM1、PXA2 在格特隐球菌耐药性中的作用。我们的研究结果证实， ATM1 基因促进格特隐球菌对抗真菌药物产生耐药性，该结果尚未报道，这一发现为临床真菌耐药性的研究提供理论基础。

Cryptococcus gattii is a strain of the yeast Cryptococcus, which is found mainly in soil, trees and tree cavities. It is a pathogenic fungus that infects immunocompetent and healthy individuals. With the global spread of Cryptococcus gattii cases of Cryptococcus gattii infection have been reported from six continents, but its sister species, Cryptococcus neoformans, has been studied more internationally and Cryptococcus gattii has been studied less. In addition, the lack of tools to study the molecular biology of Cryptococcus gattii has severely hampered the investigation of its pathogenesis. CRISPR-Cas9, an acquired immune system evolved from bacteria and archaea, has been designed as a gene editing tool for a variety of organisms, but its application in Cryptococcus gattii has not been reported. In this thesis, we have developed the first "suicide" CRISPR-Cas9 system for Cryptococcus gattii and applied it to the study of drug resistance in Cryptococcus gattii, providing a strong technical support for the subsequent investigation of the gene function and pathogenesis of Cryptococcus gattii.

The CRISPR-Cas9 system consists of a guide RNA and a Cas9 component with nuclease activity. Based on the genomic information of Cryptococcus gattii in the GenBank database, we firstly used the NCBI Blast tool to search for the U6 snRNA gene of Cryptococcus gattii and selected the promoter of U6 snRNA to activate the guide RNA expression. Using the pRH003 CRISPR-Cas9 plasmid established in our laboratory in Cryptococcus neoformans as a backbone, we used the promoter of the Cryptococcus gattii actin gene ACT1 to activate Cas9 protein expression and the promoter of the translation elongation factor TEF1 gene to activate the expression of the screening marker gene hph by means of expression frame substitution. Through enzymatic validation and DNA sequencing, we finally obtained the pBN003 CRISPR plasmid suitable for Cryptococcus gattii. After construction, we selected the reporter gene ADE2 to validate the CRISPR-Cas9 system in Cryptococcus gattii. By introducing the Cas9 target and the upstream and downstream homologous arms of the target gene into the pBN003 plasmid, we successfully obtained the ade2Δ mutant and achieved a recombinant knockout of the ADE2 gene. At the same time, we achieved targeted editing of ADE2 gene by introducing targeted mutant donor DNA. Combining the above experimental results, we successfully constructed the pBN003 CRISPR-Cas9 system capable of mediating Cryptococcus gattii genome editing with high efficiency.

The results of the previous study showed that the VGII strain of Cryptococcus gattii was more resistant to azoles than the VGI strain. In combination with transcriptome sequencing analysis, we found that the expression of ABC transporter proteins was significantly higher in strain VGII than in strain VGI. Among them, the ATM1 and PXA2 genes were candidate targets for regulating the azole sensitivity of Cryptococcus gattii. Therefore, to verify the potential biological roles of ATM1 and PXA2 transporter proteins in drug resistance in Cryptococcus gattii, we used the CRISPR-Cas9 knockdown system constructed in this thesis to analyse the biological functions of ATM1 and PXA2 genes. Phenotypic assays showed that deletion of the ATM1 gene resulted in impaired intracellular iron-sulfur cluster synthesis and a growth defect on media containing iron ions, thereby reducing cellular tolerance to oxidative stress. atm1Δ strains showed a growth defect in media containing sodium acetate. In addition, the mutant strain showed reduced cell growth on YPD medium containing 0.01% SDS, 1% Congo Red and high osmotic pressure, suggesting that the ATM1 gene plays an important role in cell wall synthesis and stabilisation. Due to the reduced adaptability of the atm1Δ strain to high temperature conditions and the smaller diameter of the pod membrane, the mutant strain was assayed for virulence using the Galleria mellonella, a common animal model of Cryptococcus. The results showed that the lethality of the atm1Δ-infected strain was significantly lower than that of the wild type, revealing an important role for ATM1 in the virulence of Cryptococcus gattii. In testing the susceptibility of antifungal drugs commonly used in the clinical treatment of cryptococcosis, we found that ATM1 deficient strains exhibited significant growth defects in media containing ketoconazole and itraconazole, especially in the presence of 5-FC, and that atm1Δ was almost completely unable to grow, a result that confirms the role of high ATM1 gene expression in promoting strain resistance, i.e. the ATM1 transporter protein is involved in cellular efflux of antifungal drugs. We sequenced the transcriptome of the 5-FC-treated ATM1 mutant strain against the wild type and showed that the genes with significant differences in expression were concentrated in six pathways: cell wall structure, membrane composition, haemoglobin binding, and iron-sulphur cluster binding, which is also consistent with the results of our phenotypic assay experiments, but the mechanism by which ATM1 deletion leads to a significant increase in susceptibility of the mutant strain to 5-FC is still under investigation. In addition to this, we have also successfully obtained knockout strains of the PXA2 gene, as verified by PCR and Southern blot experiments. However, under the current assay conditions, we have not yet found significant phenotypic changes in pxa2Δ, presumably the PXA2 protein functions under specific conditions.

In summary, in this thesis, we have constructed a CRISPR-Cas9 gene editing system for Cryptococcus gattii, which provides a powerful technology for studying the gene function and pathogenesis of Cryptococcus gattii. In addition, we have also used this system to investigate the role of the genes ATM1 and PXA2 in drug resistance in Cryptococcus gattii. Our results confirm that the ATM1 gene promotes resistance to antifungal drugs in Cryptococcus gattii, a finding that has not been reported before, and provides a theoretical basis for the study of clinical fungal drug resistance.