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

开发M-Drive：可回收的多靶点基因编辑的毛霉优化CAS9基因驱动系统电缆

• 批准号: 10727359
• 负责人: Jeffrey M. Rybak
• 金额: $ 10.35万
• 依托单位: ST. JUDE CHILDREN'S RESEARCH HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10727359
• 关键词: Acceleration; Address; Affect; Antifungal Agents; Antifungal Therapy; Applied Research; Aspergillus fumigatus; Azoles; Basic Science; CRISPR/Cas technology; Candida albicans; Cell Nucleus; Cells; Characteristics; Clinical; Communicable Diseases; Cyanogen Bromide; DNA cassette; Development; Diabetes Mellitus; Etiology; Foundations; Fungal Drug Resistance; Future; Gene Family; Genes; Genetic; Genetic Markers; Genetic Transcription; Genome; Goals; Guide RNA; Immunocompromised Host; Infection; Investigation; Knowledge; Life; Mediating; Mission; Modeling; Modernization; Molds; Molecular; Morphology; Mucor; Mucormycosis; Mycoses; National Institute of Allergy and Infectious Disease; Nature; Organism; Orthologous Gene; Outcome; Patients; Phenotype; Population; Predisposition; Public Health; Reproduction spores; Research; Septate; System; Testing; Therapeutic; Time; Work; antimicrobial resistant pathogen; efflux pump; fungus; gene drive system; genetic manipulation; genetic technology; innovation; model organism; mortality; novel; novel strategies; novel therapeutic intervention; paralogous gene; pathogen; pathogenic fungus; patient population; posaconazole; preservation; prevent; response; therapeutic development; tool

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.

项目摘要 粘膜圆形菌属是粘膜霉菌，是粘膜菌病的主要致病病原体，严重和 免疫功能低下的患者人群中威胁生命的感染。不幸的是，M。Circinelloides 对抗真菌性具有高度的内在抗药性，使临床医生在治疗时几乎没有选择 粘膜细胞增多患者。制定战略克服M. Circinelloides的关键障碍 感染是目前缺乏可用的遗传工具，因此对遗传和 其抵抗抗真菌性能力的分子基础。我们的长期目标是提高侵入性的治疗 真菌感染通过开发新的治疗策略来克服难以治疗的真菌病原体。这 该提案的总体目的是开发可回收和粘膜圆形旋转优化的Cas9基因驱动器 能够多目标基因编辑并实施M驱动系统以识别和表征的系统 特定的M. circinelloides外排泵编码基因，影响M. Circinelloides抗真菌敏感性 并代表新的遗传和分子弱点，可以在治疗上为 发展未来的抗真菌疗法。为了实现这一目标，我们将检验我们的中心假设，即1） 可以利用M. Circinelloides细胞的多核性质来实施有效且可回收的Cas9- 介导的基因驱动系统，2）响应性响应性的特定循环泵泵基因的破坏 对postaconazole治疗将对M. Circinelloides抗真菌敏感性产生影响，3）利用 M-drive系统将允许快速识别和表征这些旋转旋转的流体 影响抗真菌敏感性的泵编码基因。在AIM 1中，我们将使用M. Circinelloides pyrg-标记系统以构建M-Drive兼容（Cas9+）应变并确认功能和 通过破坏先前表征的Carrp和CNBR基因的效率 同时地。在AIM 2中，我们将实施M驱动系统，以破坏优先的一组排出泵 - 编码针对寄生虫治疗的转录上调的基因并确定 它们对对寄生虫以及其他抗真菌药物的易感性的影响。我们的方法是创新的 并包含重要的技术和概念进步，预计将对 研究粘膜菌病并对对M的遗传负债的理解产生积极影响。 Circinelloides。拟议的研究非常重要，因为完成后，我们将创建粘液 优化的遗传工具将首次成为该模型中快速有效的基因编辑 粘膜病原体。然后，这些研究将加速循环链球菌的全面表征 M. circinelloides需要抗真菌剂并建立A的基因和基因家族 制定克服粘膜菌病的治疗策略的基础。