Development of M-Drive: A recyclable Mucor-optimized CAS9 gene-drive system cable of multi-target gene editing

项目总结 卷状毛霉是一种毛霉菌，是毛霉病的主要病原体，是一种严重和 免疫受损患者群体中危及生命的感染。不幸的是，圆环杆菌 对抗真菌药物具有高度的内在耐药性，使临床医生在治疗时几乎没有选择 毛霉病患者。制定克服盘状芽胞杆菌策略的关键障碍 感染是目前缺乏可用的遗传工具，并因此缺乏对遗传和 其抵抗抗真菌药物能力的分子基础。我们的长期目标是推进侵袭性疾病的治疗 通过开发新的治疗策略来克服难以治疗的真菌病原体。这个 这项提议的总体目标是开发一种可回收的、经环状毛霉优化的Cas9基因驱动 能够进行多目标基因编辑的系统，并实现了M-Drive系统的识别和表征 影响圆环菌抗真菌敏感性的特异性圆环杆菌泵编码基因 代表了新的遗传和分子弱点，可用于治疗 未来抗真菌疗法的发展。为了实现这一点，我们将检验我们的中心假设：1) 可以利用圆环菌细胞的多核特性来实现高效和可回收的Cas9- 介导的基因驱动系统，2)破坏有反应的特定的圆环菌外排泵基因 泊沙康唑治疗会对圆环菌对真菌的敏感性产生影响，3)利用 M-Drive系统将允许快速识别和表征这些环状分枝杆菌的外排 影响抗真菌敏感性的泵编码基因。在目标1中，我们将利用我们的环状分枝杆菌 构建M-Drive相容(Cas9)菌株的PYRG标记系统，并确认其功能和 M-Drive系统通过破坏先前确定的CARRP和cnbR基因的效率 同时。在目标2中，我们将实施M-Drive系统，以扰乱一套按优先顺序排列的外流泵- 编码泊沙康唑治疗后转录上调的基因，并确定 它们对泊沙康唑和其他抗真菌药物敏感性的影响。我们的方法是创新的 并包含重要的技术和概念上的进步，预计这些进步将对 对毛霉菌病的研究，对了解毛霉菌的遗传易感性有积极的影响。 圆环状线虫。这项拟议的研究具有重要意义，因为完成后，我们将创造毛霉- 经过优化的基因工具将首次使在该模型中进行快速高效的基因编辑成为可能 毛霉病原体。这些研究将加速环状分枝杆菌的全面鉴定。 圆环菌对抗真菌药物的抗性所需的基因和基因家族 为开发克服毛霉病的治疗策略奠定了基础。

PROJECT SUMMARY Mucor circinelloides is a Mucoralean mold and a primary causative pathogen of mucormycosis, a severe and life-threatening infection among immunocompromised patient populations. Unfortunately, M. circinelloides possesses a high degree of intrinsic resistance to antifungals, leaving clinicians with few options when treating patients with mucormycosis. A critical barrier to the development of strategies to overcome M. circinelloides infections is a current lack of available genetic tools and a consequent lack of understanding of the genetic and molecular basis of its ability to resist antifungals. Our long-term goal is to advance the treatment of invasive fungal infections by developing new therapeutic strategies to overcome difficult-to-treat fungal pathogens. The overall objective of this proposal is to develop a recyclable and Mucor circinelloides-optimized Cas9 gene-drive system capable of multi-target gene-editing, and to implement the M-Drive system to identify and characterize the specific M. circinelloides efflux pump-encoding genes which impact M. circinelloides antifungal susceptibility and represent novel genetic and molecular weak-points which can be therapeutically exploited for the development of future antifungal therapies. To achieve this, we will test our central hypothesis that 1) that the multinucleate nature of M. circinelloides cells can be exploited to implement an efficient and recyclable Cas9- mediated gene-drive system, 2) disruption of specific M. circinelloides efflux pump genes which are responsive to posaconazole treatment will have an impact on M. circinelloides antifungal susceptibility, and 3) leveraging the M-Drive system will allow for the rapid identification and characterization of these M. circinelloides efflux pump-encoding genes which influence antifungal susceptibility. In Aim 1, we will utilize our M. circinelloides pyrG- marker system to construct the M-Drive compatible (Cas9+) strain and confirm the functionality and efficiency of the M-Drive system by disrupting the previously characterized carRP and cnbR genes simultaneously. In Aim 2, we will implement the M-Drive system to disrupt a prioritized set of efflux pump- encoding genes which are transcriptionally up-regulated in response to posaconazole treatment and determine their impact on susceptibility to posaconazole as well as other antifungal agents. Our approach is innovative and contains important technical and conceptual advances that are expected to contribute significantly to the study of mucormycosis and to have a positive impact on the understanding of the genetic liabilities of M. circinelloides. The proposed research is significant in that upon completion, we will have created Mucor- optimized genetic tools will make possible, for the first time, rapid and efficient gene-editing in this model Mucoralean pathogen. These studies will then accelerate the comprehensive characterization of M. circinelloides genes and gene families which are required for M. circinelloides to resist antifungal agents and establish a foundation for the development of therapeutic strategies to overcome mucormycosis.