Synergistic genetic interactions in Mycobacterium tuberculosis

Here we have constructed a pooled library of multiplexed CRISPR interference plasmids. Each plasmid expresses two distinct gRNAs that each target unique genes. In total 56 gRNAs were used in all possible pairwise combination producing >3000 possible combinations. Using the plasmid library, pooled competition experiments and next generation sequencing and we looked for gRNA combinations that had synergistic interactions. Priority combinations were validated experimentally in follow up experiments.