CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis

AbstractMycobacterium 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 96 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 prioritise potential drug targets.

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CRISPR Interference of Adenylate Cyclases from Mycobacterium tuberculosis

Applied Biochemistry and Microbiology ◽

10.1134/s0003683821040128 ◽

2021 ◽

Vol 57 (4) ◽

pp. 421-425

Author(s):

N. I. Nadolinskaia ◽

M. V. Zamakhaev ◽

M. S. Shumkov ◽

D. K. Armianinova ◽

D. S. Karpov ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Crispr Interference

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Chemical validation of Mycobacterium tuberculosis phosphopantetheine adenylyltransferase using fragment linking and CRISPR interference

10.1101/2020.09.04.280388 ◽

2020 ◽

Author(s):

Jamal El Bakali ◽

Michal Blaszczyk ◽

Joanna C. Evans ◽

Jennifer A. Boland ◽

William J. McCarthy ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Crystal Structures ◽

Active Site ◽

Drug Target ◽

Potential Drug Target ◽

Biosynthesis Pathway ◽

Potential Drug ◽

Antimicrobial Drugs ◽

X Ray ◽

Crispr Interference

AbstractThe coenzyme A (CoA) biosynthesis pathway has attracted attention as a potential target for much-needed novel antimicrobial drugs, including for the treatment of tuberculosis (TB), the lethal disease caused by Mycobacterium tuberculosis (Mtb). Seeking to identify the first inhibitors of Mtb phosphopantetheine adenylyltransferase (MtbPPAT), the enzyme that catalyses the penultimate step in CoA biosynthesis, we performed a fragment screen. In doing so, we discovered three series of fragments that occupy distinct regions of the MtbPPAT active site, presenting a unique opportunity for fragment linking. Here we show how, guided by X-ray crystal structures, we could link weakly-binding fragments to produce an active site binder with a KD < 20 μM and on-target anti-Mtb activity, as demonstrated using CRISPR interference. This study represents a big step toward validating MtbPPAT as a potential drug target and designing a MtbPPAT-targeting anti-TB drug.Abstract Figure

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Reprogramming the endogenous type III-A CRISPR-Cas system for genome editing, RNA interference and CRISPRi screening in Mycobacterium tuberculosis

10.1101/2020.03.09.983494 ◽

2020 ◽

Author(s):

Khaista Rahman ◽

Muhammad Jamal ◽

Xi Chen ◽

Wei Zhou ◽

Bin Yang ◽

...

Keyword(s):

Infectious Disease ◽

Mycobacterium Tuberculosis ◽

Rna Interference ◽

Functional Genomics ◽

Genome Editing ◽

Antibiotics Resistance ◽

Screening Methods ◽

Crispr Interference ◽

Type Iii ◽

Genome Wide

AbstractMycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of the functional genomics of M.tb and development of drugs and vaccines are hampered by the complicated and time-consuming genetic manipulation techniques for M.tb. Here, we reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene editing, RNA interference and screening via simple delivery of a plasmid harboring a mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out by this system, which was confirmed by whole-genome sequencing. This system was further employed for single and simultaneous multiple-gene RNA interference. Moreover, we successfully applied this system for genome-wide CRISPR interference screening to identify the in-vitro and intracellular growth-regulating genes. This system can be extensively used to explore the functional genomics of M.tb and facilitate the development of new anti-Mycobacterial drugs and vaccines.SummaryTuberculosis caused by Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than ten million people annually. To dissect the functional genomics and understand its virulence, persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput screening methods are desperately wanted. Our study developed an efficient and a robust tool for genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. Moreover, the system has been successfully applied for genome-wide CRISPR interference screening. This tool could be employed to explore the functional genomics of M.tb and facilitate the development of anti-M.tb drugs and vaccines.

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An Alternative, Cas12a-based CRISPR Interference System for Mycobacteria

10.1101/2021.01.25.427309 ◽

2021 ◽

Author(s):

Neil Fleck ◽

Christoph Grundner

Keyword(s):

Mycobacterium Tuberculosis ◽

Higher Order ◽

Genetic Interactions ◽

Gene Repression ◽

Crispr Interference ◽

Interference System ◽

Multiple Genes ◽

Technical Challenges

ABSTRACTThe introduction of CRISPR interference (CRISPRi) has made gene repression in mycobacteria much more efficient, but technical challenges of the prototypical Cas9-based platform, for example in multigene regulation, remain. Here, we introduce an alternative CRSPRi platform that uses the minimal Cas12a enzyme in combination with synthetic CRISPR arrays. This system is simple, tunable, and can regulate multiple genes simultaneously, providing a new tool to probe higher-order genetic interactions in mycobacteria including Mycobacterium tuberculosis (Mtb).

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Utilization of CRISPR Interference To Validate MmpL3 as a Drug Target in Mycobacterium tuberculosis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00629-19 ◽

2019 ◽

Vol 63 (8) ◽

Cited By ~ 10

Author(s):

Matthew B. McNeil ◽

Gregory M. Cook

Keyword(s):

Mycobacterium Tuberculosis ◽

Drug Discovery ◽

Mode Of Action ◽

Drug Target ◽

Content Type ◽

Crispr Interference ◽

Novel Therapeutics ◽

Novel Targets ◽

Genetic Strategies

ABSTRACT There is an urgent need for novel therapeutics to treat Mycobacterium tuberculosis infections. Genetic strategies for validating novel targets are available, yet their time-consuming nature limits their utility. Here, using MmpL3 as a model target, we report on the application of mycobacterial CRISPR interference for the rapid validation of target essentiality and compound mode of action. This strategy has the potential to rapidly accelerate tuberculosis drug discovery.

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High-resolution SEM and STEM of adrenocortical endothelium: Molecular resolution of membrane complexes

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100148885 ◽

1993 ◽

Vol 51 ◽

pp. 610-611

Author(s):

Robert P. Apkarian

Keyword(s):

Fine Structure ◽

Low Voltage ◽

Uranyl Acetate ◽

Correlative Microscopy ◽

Coated Pits ◽

Fine Grain ◽

Structural Identity ◽

Cellular Pathways ◽

Steroidogenic Cells ◽

Cr Film

A multitude of complex ultrastructural features are involved in endothelial cell (EC) gating and sorting of lipid through capillaries and into steroidogenic cells of the adrenal cortex. Correlative microscopy is necessary to distinguish the structural identity of features involved in specific cellular pathways. In addition to diaphragmed fenestrae that frequently appear in clusters, other 60-80 nm openings; plasmalemma vesicles (PV), channels and pockets fitted with diaphragms of the same dimension, coexist on the thin EC surface. Non-diaphragmed coated pits (CP) (100-120 nm) involved in receptor mediated endocytosis were also present on the EC membrane. The present study employed HRSEM of cryofractured and chromium coated specimens and low voltage HRSTEM of 80 nm thick LX-112 embedded sections stained with 2.0% uranyl acetate. Both preparations were imaged at 25 kV with a Topcon DS-130 FESEM equipped with in-lens stage and STEM detector.HRSEM images of the capillary lumen coated with a lnm continuous fine grain Cr film, provided the ability to scan many openings and resolve (SE-I contrast) the fine structure of diaphragm spokes and central densities (Fig. 1).

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Early signalling events of autophagy

Essays in Biochemistry ◽

10.1042/bse0550001 ◽

2013 ◽

Vol 55 ◽

pp. 1-15 ◽

Cited By ~ 16

Author(s):

Laura E. Gallagher ◽

Edmond Y.W. Chan

Keyword(s):

Protein Kinase ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Mammalian Cells ◽

Mammalian Target Of Rapamycin ◽

Interacting Protein ◽

The Core ◽

Autophagy Gene ◽

Autophagy Induction ◽

Cellular Pathways

Autophagy is a conserved cellular degradative process important for cellular homoeostasis and survival. An early committal step during the initiation of autophagy requires the actions of a protein kinase called ATG1 (autophagy gene 1). In mammalian cells, ATG1 is represented by ULK1 (uncoordinated-51-like kinase 1), which relies on its essential regulatory cofactors mATG13, FIP200 (focal adhesion kinase family-interacting protein 200 kDa) and ATG101. Much evidence indicates that mTORC1 [mechanistic (also known as mammalian) target of rapamycin complex 1] signals downstream to the ULK1 complex to negatively regulate autophagy. In this chapter, we discuss our understanding on how the mTORC1–ULK1 signalling axis drives the initial steps of autophagy induction. We conclude with a summary of our growing appreciation of the additional cellular pathways that interconnect with the core mTORC1–ULK1 signalling module.

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