CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

Wet, Timothy J. de; Gobe, Irene; Mhlanga, Musa M.; Warner, Digby F.

doi:10.1101/358275

Cited by 22 publications

(35 citation statements)

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“…We aimed to construct an arrayed library of inducible CRISPRi mutants ( Figure 1A ) targeting essential M. smegmatis genes ( de Wet et al, 2018 ). To this end, we leveraged an optimized mycobacterial CRISPRi system ( Rock et al, 2017 ) and previously generated genome-wide CRISPRi-Seq data ( de Wet et al, 2018 ) from which we were able to identify the set of highest efficiency single-guide (sg)RNAs targeting 294 essential M. smegmatis genes with direct M. tuberculosis homologs ( Supplementary file 1 ).…”

Section: Resultsmentioning

confidence: 99%

“…The most commonly applied functional genomics methods in bacteria – transposon-sequencing (Tn-Seq) ( van Opijnen and Camilli, 2013 ) and, increasingly, CRISPR-interference (CRISPRi)-Seq ( de Wet et al, 2018 ; Wang et al, 2018 ; Lee et al, 2019 ) – combine pooled mutagenesis with next-generation sequencing, returning quantitative estimates of fitness (via relative abundance) of mutants in a particular growth condition. Common to these approaches is that they report on the capacity of the cells to produce biomass, thereby excluding other – potentially subtler – measures of the physiological consequences of target gene disruption.…”

Section: Introductionmentioning

confidence: 99%

“…Here, we couple key recent advances in mycobacterial CRISPRi ( Rock et al, 2017 ) with quantitative microscopic imaging ( Ducret et al, 2016 ) in developing a platform enabling whole-cell characterization of essential gene knockdown in the non-pathogenic model mycobacterium, M. smegmatis . Leveraging available gene essentiality and CRISPRi guide efficacy data ( de Wet et al, 2018 ), we describe the construction of an arrayed collection of 276 validated CRISPRi mutants targeting essential M. smegmatis homologs of M. tuberculosis genes. Applying high-throughput quantitative imaging to the arrayed library, we implement a bespoke analytic pipeline to probe essential gene function at scale.…”

Section: Introductionmentioning

confidence: 99%

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Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Wet

Winkler

Mhlanga

et al. 2020

eLife

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Mycobacterium tuberculosis possesses a large number of genes of unknown or predicted function, undermining fundamental understanding of pathogenicity and drug susceptibility. To address this challenge, we developed a high-throughput functional genomics approach combining inducible CRISPR-interference and image-based analyses of morphological features and sub-cellular chromosomal localizations in the related non-pathogen, M. smegmatis. Applying automated imaging and analysis to 263 essential gene knockdown mutants in an arrayed library, we derive robust, quantitative descriptions of bacillary morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate that functionally related genes cluster by morphotypic similarity and that this information can be used to inform investigations of gene function. Exploiting this observation, we infer the existence of a mycobacterial restriction-modification system, and identify filamentation as a defining mycobacterial response to histidine starvation. Our results support the application of large-scale image-based analyses for mycobacterial functional genomics, simultaneously establishing the utility of this approach for drug mechanism-of-action studies.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Wet

Winkler

Mhlanga

et al. 2020

eLife

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“…According to a review 114 of the literature (Supplementary Table 3), approximately 70% of the strains in our library 115 lacked whole-cell phenotypic characterization in either M. tuberculosis or M. smegmatis. 116 Furthermore, almost 40% of the targeted genes and their protein products had no 117 biochemical or structural information ( Supplementary Table 3 For each gene, the highest efficiency sgRNA was identified from a previous pooled CRISPRi-Seq 122 screen (de Wet, Gobe et al 2018) and synthesized as an annealed oligonucleotide. Cloning was 123 performed at scale, followed by electroporation into an M. smegmatis ParB-mCherry reporter 124 strain (Santi and McKinney 2015).…”

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confidence: 99%

Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes

Wet

Winkler

Mhlanga

et al. 2020

Preprint

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13Mycobacterium tuberculosis possesses a large number of genes of unknown or merely 14 predicted function, undermining fundamental understanding of pathogenicity and drug 15 susceptibility. To address this challenge, we developed a high-throughput functional 16 genomics approach combining inducible CRISPR-interference and image-based analyses of 17 morphological features and sub-cellular molecular localizations in the related non-pathogen, 18M. smegmatis. Applying automated imaging and analysis to an arrayed library of 272 19 essential gene knockdown mutants, we derive robust, quantitative descriptions of bacillary 20 morphologies consequent on gene silencing. Leveraging statistical-learning, we demonstrate 21 that functionally related genes cluster by morphotypic similarity and that this information 22can be used to infer gene function. Exploiting this observation, we reveal a previously 23 unknown restriction-modification system, and identify filamentation as a defining 24 mycobacterial response to histidine starvation. Our results support the application of large-25 scale image-based analyses for mycobacterial functional genomics, simultaneously 26 establishing the utility of this approach for drug mechanism-of-action studies.

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“…Whole-genome screening via gene silencing based on the CRISPR-Cas system is a powerful approach and has been widely used to explore gene function (Rousset et al, 2018; Wang et al, 2018). Recently, Wet et al applied CRISPR-dCas9 screens for functional characterization of transcription factors in M.smegmatis (de Wet et al, 2018). However, no study has been performed on the genome scale in M.tb .…”

Section: Discussionmentioning

confidence: 99%

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

Rahman

Jamal

Chen

et al. 2020

Preprint

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Tuberculosis caused by 15 Mycobacterium tuberculosis (M.tb) is the current leading infectious disease affecting more than 16 ten million people annually. To dissect the functional genomics and understand its virulence, 17 persistence, and antibiotics resistance, a powerful genome editing tool and high-throughput 18 screening methods are desperately wanted. Our study developed an efficient and a robust tool for 19 genome editing and RNA interference in M.tb using its endogenous CRISPR cas10 system. 20 Moreover, the system has been successfully applied for genome-wide CRISPR interference 21 screening. This tool could be employed to explore the functional genomics of M.tb and facilitate 22 the development of anti-M.tb drugs and vaccines. 23 Abstract: 24 Mycobacterium tuberculosis (M.tb) causes the current leading infectious disease. Examination of 25 the functional genomics of M.tb and development of drugs and vaccines are hampered by the 26 complicated and time-consuming genetic manipulation techniques for M.tb. Here, we 27 reprogrammed M.tb endogenous type III-A CRISPR-Cas10 system for simple and efficient gene 28 editing, RNA interference and screening via simple delivery of a plasmid harboring a 29 mini-CRISPR array, thereby avoiding the introduction of exogenous proteins and minimizing 30proteotoxicity. We demonstrated that M.tb genes were efficiently and specifically knocked-in/out 31 by this system, which was confirmed by whole-genome sequencing. This system was further 32 employed for single and simultaneous multiple-gene RNA interference. Moreover, we 33 successfully applied this system for genome-wide CRISPR interference screening to identify the 34 in-vitro and intracellular growth-regulating genes. This system can be extensively used to 35 explore the functional genomics of M.tb and facilitate the development of new 36 anti-Mycobacterial drugs and vaccines.37

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CRISPRi-Seq for the Identification and Characterisation of Essential Mycobacterial Genes and Transcriptional Units

Cited by 22 publications

References 73 publications

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Arrayed CRISPRi and quantitative imaging describe the morphotypic landscape of essential mycobacterial genes

Arrayed CRISPRi and Quantitative Imaging Describe the Morphotypic Landscape of Essential Mycobacterial Genes

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

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