Figure 3. Strategy for measuring cross-resistance between drugs.

(a) Cells were mutagenized and barcoded using one of three approaches: (i) random mutagenesis and clone tracing, (ii) knockdown by CRISPRi or (iii) overexpression by CRISPRa. 10⁶ mutagenized clones or genome-wide CRISPRi/a libraries were expanded and split into replicate cultures, treated with single drugs, and DNA barcodes/sgRNAs abundance was measured by DNA sequencing. The resistance of cells to drug treatment was scored based on the degree of barcode enrichment, and cross-resistance was determined by significant enrichment in two or more drug treatments. (b) Schematic showing importance of selecting for resistance to single drugs not cocktails. Arrows: resistance is analogous to lower drug concentration and moves cells to different coordinates; cross-resistance (purple arrow) has same net effect as more penetrant single-drug resistance mutations (red, blue arrows). (c) By selecting mutations on single drugs the magnitude of the effect on each drug is known.

Figure 3—figure supplement 1. Dosing schedule and replication strategy for all three approaches taken to isolate cells resistant to single cytotoxic drugs in the R-CHOP combination.

(a) Experimental design for measuring drug resistance in DNA-barcoded Pfeiffer clones. 1 million unique mutagenized Pfeiffer cells individually tagged with DNA barcodes were expanded. Replicate flasks of cells (three per drug) were treated with 2 pulses of drug according to the dosing regimen depicted. Recovery was slowest after treatment by C or H, which induced sustained cell death for days after medium was exchanged and drug removed. The abundance of each clone was determined in the pre-treatment population and in each drug-treated replicate sample by high-throughput sequencing. (b) Experimental design for measuring drug resistance in a whole-genome collection of knockdown mutants (CRISPRi). Pfeiffer CRISPRi cells transduced with a genome-wide library of sgRNAs were treated with two-three 72 hr pulses of drug according to the dosing regimen depicted. The treatment schedule was adjusted in order to achieve 7–9 fewer population doublings than the DMSO control. The CRISPRi library contains 10 sgRNAs per gene which serve as internal replicates for each data point. (c) Experimental design for measuring drug resistance in a whole-genome collection of overexpression mutants (CRISPRa). K562 CRISPRa cells transduced with a genome-wide library of sgRNAs were treated with two 72 hr pulses of drug according to the dosing regimen depicted. The treatment schedule was adjusted in order to achieve 9–10 fewer population doublings than the DMSO control. The CRISPRa library contains 10 sgRNAs per gene which serve as internal replicates for each data point.