The multifaceted nature of CRISPR screens has propelled advancements in the field of functional genomics. Pooled CRISPR screens involve creating programmed genetic perturbations across multiple genomic sites in a pool of host cells subjected to a challenge, empowering researchers to identify genetic causes of desirable phenotypes. These genome-wide screens have been widely used in mammalian cells to discover biological mechanisms of diseases and drive the development of targeted drugs and therapeutics. Their use in non-model organisms, especially in microbes to improve bioprocessing-relevant phenotypes, has been limited. Further compounding this issue is the lack of bioinformatic algorithms for analyzing microbial screening data with high accuracy. Here, we describe the general approach and underlying principles for conducting pooled CRISPR knockout screens in non-conventional yeasts and performing downstream analysis of the screening data, while also reviewing state-of-the-art algorithms for identification of CRISPR screening outcomes. Application of pooled CRISPR screens to non-model yeasts holds considerable potential to uncover novel metabolic engineering targets and improve industrial bioproduction.

中文翻译：

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分析非常规微生物的 CRISPR 筛选

CRISPR 筛选的多方面性质推动了功能基因组学领域的进步。混合 CRISPR 筛选涉及在受到挑战的宿主细胞库中的多个基因组位点上创建程序化遗传扰动，使研究人员能够识别所需表型的遗传原因。这些全基因组筛选已广泛应用于哺乳动物细胞，以发现疾病的生物学机制并推动靶向药物和疗法的开发。它们在非模式生物中的应用，特别是在微生物中改善生物加工相关表型的应用受到限制。进一步加剧这个问题的是缺乏用于高精度分析微生物筛选数据的生物信息学算法。在这里，我们描述了在非常规酵母中进行混合 CRISPR 敲除筛选并对筛选数据进行下游分析的一般方法和基本原则，同时还回顾了用于识别 CRISPR 筛选结果的最先进算法。将 CRISPR 汇集筛选应用于非模型酵母具有发现新的代谢工程靶标和改善工业生物生产的巨大潜力。