CRISPRi–TnSeq maps genome-wide interactions between essential and non-essential genes in bacteria

Abstract

Genetic interactions identify functional connections between genes and pathways, establishing gene functions or druggable targets. Here we use CRISPRi–TnSeq, CRISPRi-mediated knockdown of essential genes alongside TnSeq-mediated knockout of non-essential genes, to map genome-wide interactions between essential and non-essential genes in Streptococcus pneumoniae. Transposon-mutant libraries constructed in 13 CRISPRi strains enabled screening of ~24,000 gene pairs. This identified 1,334 genetic interactions, including 754 negative and 580 positive interactions. Network analyses show that 17 non-essential genes pleiotropically interact with more than half the essential genes tested. Validation experiments confirmed that a 7-gene subset protects against perturbations. Furthermore, we reveal hidden redundancies that compensate for essential gene loss, relationships between cell wall synthesis, integrity and cell division, and show that CRISPRi–TnSeq identifies synthetic and suppressor-type relationships between both functionally linked and disparate genes and pathways. Importantly, in species where CRISPRi and Tn-Seq are established, CRISPRi–TnSeq should be straightforward to implement.