There is growing interest in the use of nonmodel microorganisms as hosts for biopharmaceutical manufacturing. These hosts require genomic engineering to meet clinically relevant product qualities and titers, but the adaptation of tools for editing genomes, such as CRISPR-Cas9, has been slow for poorly characterized hosts. Specifically, a lack of biochemical characterization of RNA polymerase III transcription has hindered reliable expression of guide RNAs in new hosts. Here, we present a sequencing-based strategy for the design of host-specific cassettes for modular, reliable, expression of guide RNAs. Using this strategy, we achieved up to 95% gene editing efficiency in the methylotrophic yeast Komagataella phaffii. We applied this approach for the rapid, multiplexed engineering of a complex phenotype, achieving humanized product glycosylation in two sequential steps of engineering. Reliable extension of simple gene editing tools to nonmodel manufacturing hosts will enable rapid engineering of manufacturing strains tuned for specific product profiles and potentially decrease the costs and timelines for process development.

中文翻译：

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CRISPR指导RNA的宿主信息表达，用于Komagataella phaffii中的基因组工程。

使用非模型微生物作为生物制药生产宿主的兴趣日益浓厚。这些宿主需要基因组工程来满足临床上相关的产品质量和效价，但是对于表征欠佳的宿主，用于编辑基因组的工具（如CRISPR-Cas9）的适应缓慢。具体来说，缺乏RNA聚合酶III转录的生化特性已阻碍了指导RNA在新宿主中的可靠表达。在这里，我们提出了一种基于序列的策略，用于设计宿主特异性盒体，用于模块化，可靠，指导RNA的表达。使用这种策略，我们在甲基营养型酵母嗜热菌（Komagataella phaffii）中实现了高达95％的基因编辑效率。我们将这种方法用于复杂表型的快速，多重工程，在两个连续的工程步骤中实现人性化的产品糖基化。将简单的基因编辑工具可靠地扩展到非模型制造主机，将能够快速设计针对特定产品概况调整的生产菌株，并有可能降低工艺开发的成本和时间表。