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.

遗传相互作用识别基因和通路之间的功能联系，建立基因功能或可药物靶标。在这里，我们使用 CRISPRi–TnSeq（CRISPRi 介导的必需基因敲除和 TnSeq 介导的非必需基因敲除）来绘制肺炎链球菌中必需基因和非必需基因之间的全基因组相互作用。在 13 个 CRISPRi 菌株中构建的转座子突变文库能够筛选约 24,000 个基因对。这确定了 1,334 种遗传相互作用，其中包括 754 种消极相互作用和 580 种积极相互作用。网络分析表明，17 个非必需基因与一半以上测试的必需基因存在多效性相互作用。验证实验证实，7 个基因子集可以防止扰动。此外，我们揭示了补偿必需基因丢失的隐藏冗余、细胞壁合成、完整性和细胞分裂之间的关系，并表明 CRISPRi-TnSeq 识别了功能相关和不同的基因和通路之间的合成和抑制型关系。重要的是，在建立了 CRISPRi 和 Tn-Seq 的物种中，CRISPRi–TnSeq 应该易于实施。