Combinatorial sgRNA-library preparation

To generate the pooled storage-vector library, equal amounts of the 358 single-sgRNA-expression vectors were pooled. Pooled lentiviral vector libraries containing combinatorial gRNAs were constructed with the strategy outlined in Supplementary Figure 1b. Briefly, the pooled mU6-sgRNA inserts were excised in a one-pot digestion of the pooled storage-vector library with XbaI and XhoI. The destination lentiviral vectors were digested with SpeI and SalI. The digested inserts and vectors were ligated via their compatible ends (i.e., XbaI–SpeI and XhoI–SalI) to create the pooled double-sgRNA library (358 × 358 = 128,164 total combinations) in the lentiviral vector. The lentiviral sgRNA-library pools were prepared in DH5α ultracompetent cells (Agilent Technologies) and purified with a Plasmid Midi Kit (Macherey-Nagel). The representation of each of the double-sgRNA constructs was then quantified by NGS with the oligonucleotides listed in Supplementary Table 6.