Protocol for performing pooled CRISPR-Cas9 loss-of-function screens

Alyssa J Mathiowetz; Melissa A Roberts; David W Morgens; James A Olzmann; Zhipeng Li

doi:10.1016/j.xpro.2023.102201

Protocol for performing pooled CRISPR-Cas9 loss-of-function screens

STAR Protoc. 2023 Mar 30;4(2):102201. doi: 10.1016/j.xpro.2023.102201. Online ahead of print.

Authors

Alyssa J Mathiowetz¹, Melissa A Roberts¹, David W Morgens², James A Olzmann³, Zhipeng Li⁴

Affiliations

¹ 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.
² Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA 94720, USA.
³ 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; Chan Zuckerberg Biohub, San Francisco, CA 94158, USA. Electronic address: olzmann@berkeley.edu.
⁴ 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. Electronic address: li.zhipeng@berkeley.edu.

Abstract

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),¹ Li et al. (2022),² and Roberts et al. (2022).³.

Keywords: CRISPR; Cell Culture; Cell-based Assays; Flow Cytometry/Mass Cytometry; Genomics; Molecular Biology.

Abstract

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