Figure 2 from Engineering Oncogenic Hotspot Mutations on SF3B1 via CRISPR-Directed PRECIS Mutagenesis

SF3B1 mutation-responsive reporter allows for facile isolation of SF3B1 mutant clones. A, The design of the K700E reporter. GFP is bisected by synMTERFD3i1-150 (30) and co-expressed with a blasticidin selection marker. The entire cassette is flanked by loxP signals to enable Cre recombinase-mediated deletion of reporter post-editing. B, Flow cytometry plots (left) and histograms (right) of Nalm-6 and K562 SF3B1 K700K and K700E cell lines stably transduced with the K700E reporter. C, Workflow for engineering cell lines with the K700E mutation. D, Flow cytometry plots (left) and histograms (right) of HG-3 and MEC-1 control cells or cells electroporated and sorted for PE5max K700E. E, Sanger sequencing results for HG-3 and MEC-1 control cells, cells sorted for RFP (PE5max K700E), and cells sorted for GFP (K700E reporter). F, K700E frequency measured through Sanger sequencing for each sample in biological triplicates. G, Sanger sequencing results for isogenic HG-3 and MEC-1 SF3B1 WT and K700E clones. H, Splice variant qPCR analysis of MAP3K7 and DVL2 alternative splicing in CLL, HG-3, and MEC-1 with and without SF3B1 mutation in biological triplicates. Student unpaired t-test was used (*, P ≤ 0.05; **, P ≤ 0.01). All negative controls are parental, unedited cells. Some figures created with BioRender.com.