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Competition-based proliferation assay

IM Iris Müller
AM Ann Sophie Moroni
DS Daria Shlyueva
SS Sudeep Sahadevan
ES Erwin M. Schoof
AR Aliaksandra Radzisheuskaya
JH Jonas W. Højfeldt
TT Tülin Tatar
RK Richard P. Koche
CH Chang Huang
KH Kristian Helin
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sgRNAs were designed using the CRISPR design tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design) from the Broad Institute (Supplementary Data 1, Supplementary Table 5). Each sgRNA was cloned into the U6-sgRNA-SFFV-puro-P2A-EGFP vector.

Cells expressing Cas9 were transduced with the respective sgRNA. 24 h later the media was changed, and cells were briefly selected with puromycin (1 µg/mL). Two days after transduction the cells were analyzed for GFP expression by flow cytometry and re-plated with blasticidin (10 µg/mL). At indicated time points, cells were again analyzed by flow cytometry, and re-seeded. At day 2, an aliquot was taken for extraction of genomic DNA and analysis by TIDE (https://tide.nki.nl/) (Supplementary Table 6). The gating strategy is exemplified in Supplementary Fig. 12a.

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