Journal article
Pooled CRISPR-activation screening coupled with single-cell RNA-seq in mouse embryonic stem cells
C Alda-Catalinas, MA Eckersley-Maslin, W Reik
STAR Protocols | ELSEVIER | Published : 2021
Open access
Abstract
CRISPR/Cas9 screens are a powerful approach to identify key regulators of biological processes. By combining pooled CRISPR/Cas9 screening with 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 10× Genomics scRNA-seq as a readout. For complete information on the generation and use of this protocol, please refer to Alda-Catalinas et al. (2020).
Grants
Awarded by Medical Research Council
Funding Acknowledgements
The authors thank all present and past members of the Reik and Stegle laboratories for helpful discussions. We also thank Danila Bredikhin, Oliver Stegle, and Irene Hernando-Herraez for generating code to analyze datasets that are generated as an output of this protocol. Lenti dCAS-VP64_Blast (Addgene 61425) and lenti MS2-P65-HSF1_Hygro (Addgene 61426) were a gift from Feng Zhang. pMD2.G (Addgene 12259) and psPAX2 (Addgene 12260) were a gift from Didier Trono. C.A.-C. was supported by a postgraduate award by UK Research and Innovation (UKRI, 1645504). M.A.E.-M. was supported by a BBSRC Discovery Fellowship (BB/T009713/1). Research in the Reik laboratory is supported by BBSRC (BBS/E/B/000C0422) and Wellcome Trust (105031/Z/14/Z; 210754/Z/18/Z).