Department of Chemical and Environmental Engineering, University of California, Riverside, CA, USA.
Methods Mol Biol. 2024;2760:169-198. doi: 10.1007/978-1-0716-3658-9_11.
Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.
II 类 V 型内切核酸酶越来越多地被用于开发复杂且易于使用的合成生物学工具，用于在广泛的生物体中进行基因组编辑、转录调控和功能基因组筛选。其中一种内切核酸酶 Cas12a 作为基于 Cas9 的系统的一种有吸引力的替代方案。其能够从单个转录本中成熟自身的向导 RNA（gRNA），从而实现易于多重化，并且不需要 tracrRNA 元件，也允许短 gRNA 表达盒。为了将这些功能扩展到具有工业相关性的产油酵母解脂耶氏酵母中，我们开发了一套 CRISPR-Cas12a 载体，用于方便的多重基因敲除、抑制和激活。我们进一步通过构建靶向每个基因的具有八倍覆盖率的全基因组向导库，扩展了这种 CRISPR-Cas12a 系统的功能，用于进行功能基因组筛选。使用这个文库进行的 pooled CRISPR 筛选用于分析 Cas12a 向导的活性，并开发了一种机器学习算法，可以准确预测高效的 Cas12a gRNA。在本方案章节中，我们首先介绍了一种通过 CRISPR-Cas12a 系统形成插入缺失来功能性破坏蛋白质编码基因的方法。进一步，我们描述了如何将 Cas12a 融合到转录调节剂中，并结合缩短的 gRNA 来实现转录抑制或激活。最后，我们描述了全基因组文库的设计、克隆和验证，以及执行 pooled CRISPR 筛选的方案，以确定在 Y. lipolytica 中全基因组范围内的向导活性图谱。这里讨论的工具和策略扩展了在这个重要工业宿主中进行简便基因组工程的可用合成生物学工具列表。
