ll OPEN ACCESS Protocol Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells Celia Alda-Catalinas, Melanie A. Eckersley-Maslin, Wolf Reik celia.x.aldacatalinas@gsk. com (C.A.-C.)
[email protected]. uk (W.R.) Highlights Protocol for CRISPRa screens with single- cell readout to interrogate gene function Detailed description of CRISPRa screening procedures in mouse embryonic stem cells Detailed steps on how to construct derived single-cell sgRNA amplicon libraries CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with a single-cell RNA-sequencing readout, individual perturbations can be assessed in parallel both comprehensively and at scale. Importantly, this allows gene function and regulation to be interrogated at a cellular level in an unbiased manner. Here, we present a protocol to perform pooled CRISPR-activation screens in mouse embryonic stem cells using 103 Genomics scRNA-seq as a readout. Alda-Catalinas et al., STAR Protocols 2, 100426 June 18, 2021 ª 2021 The Authors. https://doi.org/10.1016/ j.xpro.2021.100426 ll OPEN ACCESS Protocol Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells Celia Alda-Catalinas,1,4,7,* Melanie A. Eckersley-Maslin,1,5,6 and Wolf Reik1,2,3,8,* 1Epigenetics Programme, Babraham Institute, Cambridge CB22 3AT, UK 2Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK 3Centre for Trophoblast Research, University of