Efficient CRISPR/Cas9-mediated gene editing to mutate genes that encode the prosurvival BCL2 proteins. (A) The OPM-2 cell line relies on MCL1 for its survival. The viability of OPM-2 cells 72 hours after addition of doxycycline (DOX) to induce expression of sgRNAs that target BCL2, BCLXL, BCLW, MCL1, or BCL2A1 was determined with CellTiter-Glo assays.22 Two sgRNAs were tested for each gene: sgRNA#1 (darker shade) and sg#2 (lighter shade). The panel on the right shows the viability of cells expressing the empty vector (EV; control) or MCL1 sgRNA#1 0 to 96 hours after addition of DOX to induce expression of the guide RNAs. (B) Caspases mediate the killing of OPM-2 cells when MCL1 is genetically targeted. The proportion of dying cells (PI uptake determined by flow cytometry) of OPM-2 cells 24 hours after switching on the expression of the sgRNA (+ DOX) to target MCL1 was determined when cultured with (+) or without (−) the broad-spectrum caspase inhibitor Q-VD-OPh (25 μM). Data (means ± 1 SD) are derived from 2 independent experiments. (C) KMS-12-PE cells rapidly lose their viability when BCL2 or MCL1 is genetically ablated. Similar experiments to that in A, but undertaken with KMS-12-PE cells. Although genetically targeting BCLXL, BCLW, or BCL2A1 has no effect, targeting BCL2 or MCL1 readily kills KMS-12-PE cells. (D) Deleting BCL2, BCLXL, BCLW, MCL1, or BCL2A1 had no effect on the viability of U266B1 cells. Similar experiments to that in A and C, but undertaken with U266B1 cells. (E) U266B1 cells rely on MCL1 and BCLXL for their survival. The sensitivity (in IC50s) of U266B1 cells to selective inhibition of BCL2 (with ABT-199) or BCLXL (with A5463) was determined at 48 hours when MCL1 was genetically targeted (+DOX) or not (−DOX). Of note, the concomitant targeting of BCLXL (pharmacologically) and MCL1 (genetically) readily killed U266B1 cells, whereas the cotargeting of BCL2 and MCL1 did not. (F) Ablating BCL2, BCLXL, BCLW, or MCL1 protein expression. Immunoblotting of cell lysates prepared from pools of the human myeloma cell line U266B1 after DOX addition for 72 hours to induce expression of sgRNAs that target expression of the indicated proteins. Compared with the control cells expressing the empty vector (EV; left lanes), the levels of BCL2, BCLXL, BCLW, or MCL1 are significantly reduced in the pools analyzed. HSP70, loading control. (G) Efficient deletion of BCL2A1. Genomic DNA prepared from U266B1 expressing an sgRNA to BCL2A1 (sgRNA #1) was subjected to Sanger DNA sequencing. The table shows the top 10 sequencing reads; 94% of all the reads (3902 in total) were indels. Of these, the majority is predicted to be deleterious to expression of BCL2A1 by causing frame shift mutations (red in the pie chart); some were in frame indels (beige). Red dashes, deleted bases; red letters, insertions or substitutions. The sequencing data for all the sgRNAs used in our study, including those of the cells shown in panel D, as well as their characterization, are shown in supplemental Figure 5-13. The results in panels A, C, D, and E represent the data from representative experiments performed in triplicates and shown as means ± 1 SD.
