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， 具有免疫缺陷的个体，例如具有中性粒细胞减少症或糖尿病酮症酸中毒的个体。死亡率 真菌病的发病率非常高，即使经过适当治疗，死亡率也接近50%。由于 糖尿病酮症酸中毒、癌症和器官移植的患病率增加， 风险急剧增加，例如最近医院获得性根霉病爆发的情况 新奥尔良的delemar感染导致至少6名儿童死亡。尽管有这样 重要的是，非常有限的研究资源可用于研究R。delemar发病机制。这是 部分原因是该生物体不像其他致病真菌那样易于遗传。比如说， 由于全基因组复制，通常存在多个基因拷贝，很少有有丝分裂稳定的遗传 可以用现有的方法获得转化体。我们建议开发CRISPR-Cas （重复的规则间隔短回文重复序列-CRISPR相关）系统， 高效的基因特异性操作和定制的基因组编辑。delemar。我们将开始 与原型菌株相比，表征独特临床菌株的相关特征， 致病性、营养缺陷型和抗真菌化合物抗性。我们将构建Cas9的载体 使用内源启动子和终止子序列的核酸酶和指导RNA（gRNA）表达。 然后，我们将通过操纵标记PYRF（URA 5）来测试CRISPR-Cas系统的效用。 编码乳清酸核苷磷酸脱羧酶的基因，以及编码 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、耐药性和发病机制。我们的研究计划的成功实施将 为R. delemar发病机制。

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.