Towards a genome-wide CRISPR/Cas9 mutant library in Rhizopus delemar

德尔玛根霉是威胁生命的毛霉病的主要原因 负责 约70%的临床病例，总死亡率超过50%。尽管它的 对人类健康造成毁灭性的影响， 德尔马氏杆菌的大部分病理生物学和分子疾病 机制仍不清楚。作为构建高通量全基因组 的突变体文库，我们建议 利用以下技术生成小规模靶向突变体文库 多路复用CRISPR/CAS9技术。在具体目标1中，我们将综合一组单一指南 针对40个随机选择的基因的RNA(SgRNAs)，并将汇集的sgRNAs和Cas9合二为一- 将载体构建到R.delemar。在特定目标2中，我们将使用Sanger DNA识别基因突变 测序和高通量下一代测序。我们还将评估疗效、特异性、 和多重CRISPR/Cas9诱发的靶外突变。这项探索性研究的成功 这项工作将导致构建全基因组CRISPR/Cas9突变文库，用于系统 德尔玛黄粉菌致病机理的研究。

Rhizopus delemar is the primary cause of life-threatening mucormycosis responsible for approximately 70% of clinical cases, with an overall mortality rate exceeding 50%. Despite its devastating impact on human health, much of R. delemar’s pathobiology and molecular disease mechanisms remains unknown. As an initial step towards constructing a high-throughput genome-wide mutant library of R. delemar, we propose to generate a small-scale targeted mutant library utilizing multiplex CRISPR/Cas9 technology. In Specific Aim 1, we will synthesize a collection of single-guide RNAs (sgRNAs) targeting 40 randomly selected genes and deliver pooled sgRNAs and Cas9 in a one- vector construct into R. delemar. In Specific Aim 2, we will identify gene mutations using Sanger DNA sequencing and high-throughput next-generation sequencing. We will also assess efficacy, specificity, and off-target mutations induced by multiplex CRISPR/Cas9. The success of this exploratory research effort will lead to the construction of whole-genome CRISPR/Cas9 mutant libraries for the systemic examination of R. delemar pathogenesis mechanisms.