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

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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.