Oral Presentation Multi-Omics Inaugural Conference 2022

Pooled CRISPR activation screens with single-cell RNA-sequencing readout using CROPseq (#15)

Johannes W Wichmann 1 2 , Amali Cooray 1 2 , Gaoyuan Wang 1 2 , Sarah Diepstraten 1 2 , Valentina Snetkova 3 , Jean-Philippe Fortin 3 , Benjamin Haley 3 , Gemma L Kelly 1 2 , Marco J Herold 1 2
  1. Department of Medical Biology, University of Melbourne, Melbourne, Victoria, Australia
  2. Walter and Eliza Hall Institute of Medical Research, Parkville, VIC, Australia
  3. Department of Molecular Biology, Genentech, Inc., South San Francisco, California, USA

CROPseq1,2 is the next evolution in pooled CRISPR screens. Conventional CRISPR screens can mainly assay for simple phenotypes, such as cellular viability. The advances in single-cell RNA-sequencing and the parallel readout of CRISPR guide RNA and transcriptome on a single-cell level allows for a much richer phenotypic readout while maintaining high scalability.

We utilized cells derived from a newly developed CRISPR activation mouse3 combined with 2 small CRISPR CROPseq libraries containing 216 and 287 guide RNAs to address their suitability for in vitro and in vivo approaches. We employed two methods to capture CRISPR guides within single cells: I) capture of poly-adenylated CROPseq transcripts in 10xG 3’-tag scRNAseq and II) direct sgRNA capture using reverse transcription primer in 10xG 5’-tag scRNAseq. Additionally, we combined our pooled CRISPR screen with hashtag multiplexing and protein detection via CITE-seq.

We introduced the first CRISPR library into a cell line and selected for drug resistance prior to scRNAseq. We were able to detect sgRNAs in 93% of cells and 3689 genes per cell. Next, we reconstituted irradiated mice with embryonic CRISPR activation enabled HSCs transduced with the second CROPseq library and analysed peripheral blood, bone marrow and spleen samples. In vivo, only a portion of analysed cells contained sgRNAs of which 96% of cells were confidently assigned at least one sgRNA in whole blood together with 1749 genes per cell.

We observed high clonality within in vivo samples after HSCs implantation and continue to work on improved HSCs transduction protocols to increase sgRNA heterogeneity to exploit the full potential of our CROPseq CRISPR activation approach.

  1. Datlinger, P., et al. Pooled CRISPR screening with single-cell transcriptome readout. Nat Methods 14, 297–301 (2017). https://doi.org/10.1038/nmeth.4177
  2. Hill, A., et al. On the design of CRISPR-based single-cell molecular screens. Nat Methods 15, 271–274 (2018). https://doi.org/10.1038/nmeth.4604
  3. Deng, Y., et al. Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance. Nat Commun 13, 4739 (2022). https://doi.org/10.1038/s41467-022-32485-9