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

Abstract

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 <u>mu</u>ltiplex, <u>r</u>andomized <u>C</u>RISPR <u>i</u>nterference <u>s</u>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 44 <i>Legionella pneumophila</i> virulence genes encoding highly conserved transmembrane proteins for their role in pathogenesis. Both amoeba and human macrophages were challenged with <i>L. pneumophila</i> bearing the pooled CRISPR array libraries, leading to the identification of several new virulence-critical combinations of genes. <i>lpg2888</i> and <i>lpg3000</i> were particularly fascinating for their apparent redundant functions during <i>L. pneumophila</i> human macrophage infection, while <i>lpg3000</i> alone was essential for <i>L. pneumophila</i> virulence in the amoeban host <i>Acanthamoeba castellanii</i>. 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.