scholarly journals CRISPR Interference of Adenylate Cyclases from Mycobacterium tuberculosis

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

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

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

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

2021 ◽  
Author(s):  
Matthew B. McNeil ◽  
Laura M Keighley ◽  
Josephine R. Cook ◽  
Chen-Yi Cheung ◽  
Gregory M. Cook
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Drug Targets ◽  
Target Genes ◽  
Acid Synthesis ◽  
Essential Genes ◽  
New Drugs ◽  
Cellular Functions ◽  
Amino Acid Synthesis ◽  
Crispr Interference ◽  
Cellular Pathways

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.

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

2021 ◽  
Author(s):  
Matthew B. McNeil ◽  
Laura M Keighley ◽  
Josephine R. Cook ◽  
Chen‐Yi Cheung ◽  
Gregory M. Cook
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Crispr Interference ◽  
Cellular Pathways

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

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.

scholarly journals An Alternative, Cas12a-based CRISPR Interference System for Mycobacteria

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).

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

2019 ◽  
Vol 63 (8) ◽  
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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