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

项目摘要 卷状毛霉是毛霉属霉菌，是毛霉病的主要病原体， 免疫功能低下患者人群中危及生命的感染。不幸的是M枝毛霉 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 毛霉菌病患者。一个关键的障碍，发展战略，以克服M。枝毛霉 感染是目前缺乏可用的遗传工具，因此缺乏对遗传和 分子基础的能力，以抵抗抗真菌药。我们的长期目标是推进侵入性 通过开发新的治疗策略来克服难以治疗的真菌病原体来治疗真菌感染。的 该提案的总体目标是开发可回收的和卷枝毛霉优化的Cas9基因驱动 能够进行多靶基因编辑的系统，并实施M-Drive系统来识别和表征 特异性M. circinelloides外排泵编码基因影响M.卷枝霉属真菌敏感性 并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，我们将测试我们的中心假设，1）， M.可以利用circinelloides细胞来实现有效和可回收的Cas9- 介导的基因驱动系统; 2）特异性M. circinelloides外排泵基因， 泊沙康唑治疗对M.卷枝藻抗真菌敏感性，和3）利用 M-Drive系统将允许快速识别和表征这些M。卷丝藻外排 影响抗真菌药敏感性的泵编码基因。在目标1中，我们将使用M。枝毛霉 pyrG-标记系统以构建M-Drive相容性（Cas9+）菌株并确认功能性， 通过破坏先前表征的carRP和cnbR基因来提高M-Drive系统的效率 同步在目标2中，我们将实施M驱动系统，以破坏一组优先的外排泵- 编码响应泊沙康唑治疗而转录上调的基因， 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进步，预计将大大有助于 毛霉菌病的研究，并有积极的影响，了解遗传负债的毛霉菌。 卷枝藻这项拟议的研究意义重大，因为完成后，我们将创造毛霉- 优化的遗传工具将首次使这种模式中快速有效的基因编辑成为可能 毛霉病原体。这些研究将加速M.枝毛霉 基因和基因家族，M. circinelloides抵抗抗真菌剂，并建立一个 为开发治疗策略以克服毛霉菌病奠定了基础。

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.