Mathiowetz Alyssa J, Roberts Melissa A, Morgens David W, Olzmann James A, Li Zhipeng
Departments of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA 94720, USA; Department of Nutritional Sciences and Toxicology, University of California, Berkeley, Berkeley, CA 94720, USA.
Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
STAR Protoc. 2023 Mar 30;4(2):102201. doi: 10.1016/j.xpro.2023.102201.
Phenotypic screens involving pooled CRISPR-Cas9 libraries offer a powerful, rapid yet affordable approach to evaluate gene functions on a global scale. Here, we present a protocol for performing pooled CRISPR-Cas9 loss-of-function screens to identify genetic modifiers using either fluorescence-based or cell death phenotypic readouts. We describe steps for designing and amplifying the library and generating and screening cells. We then detail deep sequencing and statistical analysis using cas9 High Throughput maximum Likelihood Estimator. For complete details on the use and execution of this protocol, please refer to Bersuker et al. (2019), Li et al. (2022), and Roberts et al. (2022)..
涉及CRISPR-Cas9文库的表型筛选提供了一种强大、快速且经济实惠的方法，可在全球范围内评估基因功能。在此，我们展示了一种使用基于荧光或细胞死亡表型读数进行CRISPR-Cas9功能缺失筛选以鉴定基因修饰因子的方案。我们描述了文库设计、扩增以及细胞生成和筛选的步骤。然后，我们详细介绍了使用cas9高通量最大似然估计器进行深度测序和统计分析的方法。有关本方案使用和执行的完整详细信息，请参考Bersuker等人（2019年）、Li等人（2022年）和Roberts等人（2022年）的研究。
