CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis
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Evaluation Summary:
High-throughput approaches that accurately assess drug target vulberbility in Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently needed to develop new treatment options for this dreaded disease. This paper applies a CRISPRi based approach to investigate gene essentiality and vulnerability on a diverse set of 96 genes. While the key observations of the study support previous findings, the approach reported here is useful for identification and characterization of novel drug targets. The study will be of interest to microbiologists and those interested in diverse aspects of bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Abstract
Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high‐throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality, and scalability as a molecular tool. Using mycobacterial CRISPR interference in combination with phenotypic screening, we have overcome these individual issues to investigate essentiality, vulnerability and lethality for 94 target genes from a diverse array of cellular pathways, many of which are potential antibiotic targets. Essential genes involved in cell wall synthesis and central cellular functions were equally vulnerable and often had bactericidal consequences. Conversely, essential genes involved in metabolism, oxidative phosphorylation, or amino acid synthesis were less vulnerable to inhibition and frequently bacteriostatic. In conclusion, this study provides novel insights into mycobacterial genetics and biology that will help to prioritize potential drug targets.
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Evaluation Summary:
High-throughput approaches that accurately assess drug target vulberbility in Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently needed to develop new treatment options for this dreaded disease. This paper applies a CRISPRi based approach to investigate gene essentiality and vulnerability on a diverse set of 96 genes. While the key observations of the study support previous findings, the approach reported here is useful for identification and characterization of novel drug targets. The study will be of interest to microbiologists and those interested in diverse aspects of bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed …
Evaluation Summary:
High-throughput approaches that accurately assess drug target vulberbility in Mycobacterium tuberculosis, the causative agent of tuberculosis, are urgently needed to develop new treatment options for this dreaded disease. This paper applies a CRISPRi based approach to investigate gene essentiality and vulnerability on a diverse set of 96 genes. While the key observations of the study support previous findings, the approach reported here is useful for identification and characterization of novel drug targets. The study will be of interest to microbiologists and those interested in diverse aspects of bacterial metabolism.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Studies evaluating vulnerability of specific biological pathways and validation of gene essentiality are extremely slow and technically challenging in M. tuberculosis. In this study, McNeil et al employ a previously reported CRISPRi approach to evaluate gene essentiality and lethality in a high-throughput manner. They design and test sgRNA sequences against 96 genes targeting various cellular pathways. Most of the targets involved in cell wall biosynthesis or core cellular functions were found to be not only essential but gave a bactericidal phenotype when inhibited. On the other hand, the genes involved in metabolic processes although mostly essential, gave only a bacteriostatic phenotype when inhibited. This was attributed to be due to metabolic buffering upon inhibition of bacterial metabolic processes. …
Reviewer #1 (Public Review):
Studies evaluating vulnerability of specific biological pathways and validation of gene essentiality are extremely slow and technically challenging in M. tuberculosis. In this study, McNeil et al employ a previously reported CRISPRi approach to evaluate gene essentiality and lethality in a high-throughput manner. They design and test sgRNA sequences against 96 genes targeting various cellular pathways. Most of the targets involved in cell wall biosynthesis or core cellular functions were found to be not only essential but gave a bactericidal phenotype when inhibited. On the other hand, the genes involved in metabolic processes although mostly essential, gave only a bacteriostatic phenotype when inhibited. This was attributed to be due to metabolic buffering upon inhibition of bacterial metabolic processes. The authors have performed an comprehensive evaluation of their CRISPRi using this 96-set of target genes. While most of their conclusions are in line with previous observations from other gene-essentiality studies, the approach could be very useful for TB researchers.
Overall, this is a very elegant study and the manuscript is written clearly. I especially would like to commend the authors for the effort they have invested in presenting the vulnerability data in a simple and clear manner.
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Reviewer #2 (Public Review):
This work exploited a previously reported tool (CRISPRi) to repress the expression of 96 essential genes associated with diverse biological functions and in parallel-assessed the strains' fitness. The authors earlier published similar work to show the effect of transcriptionally repressing mmpL8 and ATP synthase on the growth of Mtb. Here, the authors developed a work-flow of how CRISPRi can be multiplexed in a 96 well plate format to assess bacterial growth phenotypes. A gradient of ATc concentration mediated the transcriptional repression, and CFU and OD monitored the effect on growth. By comparing OD and CFU with a no-ATc control, the authors categorized genes into essential, non-essential, and growth-impaired. Similar to earlier observations, this work confirmed that genes essential for cell wall …
Reviewer #2 (Public Review):
This work exploited a previously reported tool (CRISPRi) to repress the expression of 96 essential genes associated with diverse biological functions and in parallel-assessed the strains' fitness. The authors earlier published similar work to show the effect of transcriptionally repressing mmpL8 and ATP synthase on the growth of Mtb. Here, the authors developed a work-flow of how CRISPRi can be multiplexed in a 96 well plate format to assess bacterial growth phenotypes. A gradient of ATc concentration mediated the transcriptional repression, and CFU and OD monitored the effect on growth. By comparing OD and CFU with a no-ATc control, the authors categorized genes into essential, non-essential, and growth-impaired. Similar to earlier observations, this work confirmed that genes essential for cell wall biosynthesis and central metabolism are more vulnerable and showed bactericidal consequences. Also, since Mtb displays plasticity in respiration (Beites et al., Nature communications, 2019), essential genes involved in oxidative phosphorylation were found to be less vulnerable.
Strength
Antibacterial drug development suffers from a paucity of targets whose inhibition can selectively and quickly kill Mtb. To do this, we urgently need simple tools, which are amenable to high throughput formats. Authors describe the ease by which CRISPRi allows studying multiple target genes in parallel.
Weakness
My primary concern is that many extrapolations are made based on the assumption that transcriptional silencing's kinetics will bring about proportional changes at the protein level. For example, classifying phenotypes such as weak bactericidal, strong bactericidal, essential bacteriostatic, and growth impairing are entirely based on growth changes (OD and CFU) as a function of ATc concentration without actually measuring transcript and protein levels. There are several examples of discordance between transcript and protein levels, which affect growth phenotypes. For example, transcriptional repression of BioA reduced protein levels 72 h post-addition of ATc. It did not affect growth, whereas targeting BioA protein using the Bio-SspB/DUC switch depleted protein faster and abolished Mtb's growth (Kim et al., PNAS).
Similarly, studies have shown that growth defects do not correlate with the degree of protein depletion (Wei et al., PNAS). Only 20% depletion of RpoB arrest growth, whereas near 100% depletion of DHFR (folate metabolism) and Alr only exert a modest effect on growth. This could be due to high intracellular concentrations of DHFR and Alr. Agreeing to this, the authors also found that transcriptional repression of folate pathway (folA, folP1) and alr does not affect the viability of Mtb. Notwithstanding these observations, folate pathway and Alr are attractive drug targets.
Several discrepancies with previous studies are quite evident in this paper. Previously defined essential genes display no growth phenotype upon transcriptional repression in this study (e.g., def, rho, birA, fum, etc). Not all of these can be explained by a low PAM score or metabolic buffering.
Overall, the authors' approach is unlikely to be useful in cases where drug targets are natively expressed at a much higher level and maintain stability over a long time. Also, transcriptional approaches are inadequate to identify vulnerable targets. They suffer from leakiness, slow/partial depletion of the gene product, post-translational modifications of the target, mutations that obstruct regulation, or an amalgamation of these problems.
Minor issues
In the work-flow figure (Fig 1), why the growth of -ve control is represented lower than the growth of non- essential gene?
Instead of no-ATC control, a scrambled sgRNA control is better.
Line 55, page 4: the word "only" is appearing twice.
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