A randomized multiplex CRISPRi-Seq approach for the identification of critical combinations of genes

Identifying virulence-critical genes from pathogens is often limited by functional redundancy. To rapidly interrogate the contributions of combinations of genes to a biological outcome, we have developed a<underline>mu</underline>ltiplex, randomized<underline>C</underline>RISPR interference<underline>s</underline>equencing (MuRCiS) approach. At its center is a new method for the randomized self-assembly of CRISPR arrays from synthetic oligonucleotide pairs. When paired with PacBio long-read sequencing, MuRCiS allowed for near-comprehensive interrogation of all pairwise combinations of a group of 44Legionella pneumophilavirulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged withL. pneumophilabearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes.lpg2888andlpg3000were particularly fascinating for their apparent redundant functions duringL. pneumophilahuman macrophage infection, whilelpg3000alone was essential forL. pneumophilavirulence in the amoeban hostAcanthamoeba castellanii. Thus, MuRCiS provides a method for rapid genetic examination of even large groups of redundant genes, setting the stage for application of this technology to a variety of biological contexts and organisms.