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.