CRISPR-based high-throughput genome-wide loss-of-function screens are a valuable approach to functional genetics and strain engineering. The yeast Komagataella phaffii is a host of particular interest in the biopharmaceutical industry and as a metabolic engineering host for proteins and metabolites. Here, we design and validate a highly active 6-fold coverage genome-wide sgRNA library for this biotechnologically important yeast containing 30,848 active sgRNAs targeting over 99% of its coding sequences. Conducting fitness screens in the absence of functional non-homologous end joining (NHEJ), the dominant DNA repair mechanism in K. phaffii, provides a quantitative means to assess the activity of each sgRNA in the library. This approach allows for the experimental validation of each guide's targeting activity, leading to more precise screening outcomes. We used this approach to conduct growth screens with glucose as the sole carbon source and identify essential genes. Comparative analysis of the called gene sets identified a core set of K. phaffii essential genes, many of which relate to metabolic engineering targets, including protein production, secretion, and glycosylation. The high activity, genome-wide CRISPR library developed here enables functional genomic screening in K. phaffii, applied here to gene essentiality classification, and promises to enable other genetic screens.

中文翻译：

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由高活性 CRISPR-Cas9 文库实现的 Komagataella phaffii 中的功能基因组筛选


基于 CRISPR 的高通量全基因组功能丧失筛选是功能遗传学和菌株工程的一种有价值的方法。酵母 Komagataella phaffii 在生物制药行业中受到特别关注，并且是蛋白质和代谢物的代谢工程宿主。在这里，我们为这种生物技术上重要的酵母设计并验证了一个高活性的 6 倍覆盖全基因组 sgRNA 文库，该酵母含有 30,848 个活性 sgRNA，靶向其 99% 以上的编码序列。在没有功能性非同源末端连接 （NHEJ） 的情况下进行适应性筛选，这是 K. phaffii 中的主要 DNA 修复机制，提供了一种定量方法来评估文库中每个 sgRNA 的活性。此方法允许对每个指南的定位活动进行实验验证，从而获得更精确的筛选结果。我们使用这种方法以葡萄糖作为唯一的碳源进行生长筛选，并鉴定必需基因。对所谓基因集的比较分析确定了一组核心的 K. phaffii 必需基因，其中许多与代谢工程靶标有关，包括蛋白质产生、分泌和糖基化。这里开发的高活性、全基因组 CRISPR 文库能够在 K. phaffii 中进行功能基因组筛选，在这里应用于基因必要性分类，并有望实现其他遗传筛选。