Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes

Abstract

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions suggesting that regimens with multiple bioenergetic inhibitors will drastically shorten treatment times. However, realizing this potential is hampered by a lack of on-target inhibitors and a poor understanding of which inhibitor combinations have the strongest interactions. To overcome these limitations, we have used CRISPR interference (CRISPRi) to characterize the consequences of transcriptionally inhibiting individual respiratory complexes and identify bioenergetic complexes that when simultaneously inhibited result in cell death. In this study, we identified known and novel synthetic lethal interactions and demonstrate how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.

CRISPR-Based Approaches for Gene Regulation in Non-Model Bacteria

“…CRISPRi is particularly useful for investigating essential genes because its repression can be titrated to prevent full knockdown and cell death (Knoot et al, 2020;Bosch et al, 2021). Additionally, epistatic effects of multiple genes can easily be investigation by simply expressing multiple gRNA within the same cell (Ellis et al, 2021;McNeil et al, 2022). Although not as common as CRISPRi due to stricter design rules (Fontana et al, 2020a), CRISPRa can be used to induce expression of silent genes to investigate their functions and products, including entire silent biosynthetic gene clusters (Ke et al, 2021).…”