scRNA-seq for 117 RUNX1 variants including WT and LOF controls

Here, we deploy SEUSS, a Perturb-seq method, to generate mutations on RUNX1 and measure the impact of 115 RUNX1 Runt domain missense mutations, including positive and negative controls, in single myelogenous leukemia cells (K562). We generated a clonal K562 cell line with doxycycline-inducible CRISPRi knockdown of endogenous RUNX1 (~70% reduction). The RUNX1 variant ORF overexpression library was generated from a modified lentiviral vector to contain the RUNX1 variant (WT, mutated, or GFP as LOF control) and a 12 base pair barcode sequence unique to each variant for identification after single-cell transcriptome sequencing. The cells were transduced with the pooled variant library at a low MOI (~0.3) to ensure that each cell received a single construct and then were grown with hygromycin to select those carrying constructs. Cells were split into two populations, one treated with doxycycline to induce repression of endogenous RUNX1 (dox), the other not (nodox). The screen was conducted with two biological replicates with greater than 1 million cells in each condition to ensure greater than 1000-fold coverage of the library. At day 7 post transduction, single-cell RNA libraries were prepared and sequenced using the 10X Genomics Chromium platform.