Genome editing in Rhizopus delemar using using CRISPR-Cas systems

接合菌病是第三种最常见的侵袭性真菌感染，在 有免疫缺陷的人，如中性粒细胞减少症或糖尿病酮症酸中毒。到期死亡率 接合霉菌病的发病率非常高，即使在适当的治疗下也有近50%的死亡率。由于 糖尿病酮症酸中毒、癌症和器官移植的患病率不断上升， 风险正在急剧增加，例如最近爆发的医院获得性根霉病 新奥尔良的Delemar感染，导致至少6名儿童死亡。尽管如此 重要的是，现有的研究资源非常有限，用来研究松材线虫的发病机制。这是 部分原因是这种生物体不像其他病原真菌那样在遗传上容易驯化。例如, 由于全基因组复制，经常存在多个基因拷贝，很少有有丝分裂稳定的遗传 用现有的方法可以获得转化子。我们建议开发一个CRISPR-CAS (集群规则间隔短回文重复-CRISPR关联)系统，允许 德尔玛红豆杉的高效基因特异性操作和定制基因组编辑。我们将从 将独特的临床菌株与典型菌株进行比较，以确定与 致病性、营养缺陷性和对抗真菌化合物的抗性。我们将为Cas9构造向量 核酸酶和引导RNA(GRNA)利用内源启动子和终止子序列表达。 然后，我们将通过操纵标记PYRF(URA5)来测试CRISPR-CAS系统的效用 编码磷酸鸟苷脱羧酶的基因以及编码CNBR和CNA(A-C)的基因 钙调神经磷酸酶的调节和催化亚基，其同系物在真菌生长中起关键作用， 形态发生、耐药和发病机制。我们研究计划的成功实施将 为进一步研究松材线虫致病机理提供了一种革命性的工具。

Zygomycosis is the third most common invasive fungal infection that causes devastating diseases in individuals with immune deficiency, such as those with neutropenia or diabetic ketoacidosis. Mortality due to zygomycosis is very high, with a nearly 50% fatality rate even when appropriately treated. Due to the increasing prevalence of diabetic ketoacidosis, cancer, and organ transplantation, the number of patients at risk is dramatically increasing, such as in the case of a recent outbreak of hospital acquired Rhizopus delemar infection in New Orleans, which resulted in the death of at least six children. Despite such importance, very limited research resources are available to investigate R. delemar pathogenesis. This is partially due to the organism being not as genetically tractable as other pathogenic fungi. For example, there often exist multiple gene copies due to whole-genome duplication, and few mitotically stable genetic transformants can be obtained with the existing methodology. We propose to develop a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) system that allows efficient gene specific manipulation and custom genome editing in R. delemar. We will begin by characterizing unique clinical strains in comparison to the archetypical strain for characteristics relevant to pathogenicity, auxotrophy, and resistance to antifungal compounds. We will construct vectors for Cas9 nuclease and guide RNA (gRNA) expression using the endogenous promoter and terminator sequences. We will then test the utility of the CRISPR-Cas system through manipulation of the marker PYRF (URA5) gene encoding orotidine phosphate decarboxylase, and the CNBR and CNA(A-C) genes encoding regulatory and catalytic subunits of calcineurin whose homologs play critical roles in fungal growth, morphogenesis, drug resistance, and pathogenesis. Successful implementation of our research plan will provide a revolutionary tool in promoting genetic studies of R. delemar pathogenesis mechanism.