Strategies of genome editing in mycobacteria: Achievements and challenges

Choudhary, Eira; Lunge, Ajitesh; Agarwal, Nisheeth

doi:10.1016/j.tube.2016.03.005

Cited by 17 publications

(12 citation statements)

References 61 publications

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“…In addition to high efficiency, this system has the advantage of a simple procedure, requiring only two-step cloning (first of the gRNA and then of the cognate donor DNA). In this regard, the currently, used gene editing tool in M.tb- based phage transduction is laborious and time consuming due to the multiple cloning steps involved and the screening for positive phages; in addition this tool requires expensive kits and expertise (Bardarov et al, 2002; Choudhary et al, 2016).…”

Section: Discussionmentioning

confidence: 99%

“…The conventional methods for gene editing based on simple homologous recombination using tools such as non-replicating plasmids (Husson et al, 1990), incompatible plasmids (Pashley et al, 2003), linear DNA substrates (Balasubramanian et al, 1996) and phage-based transduction (Bardarov et al, 2002) are less efficient. Although phage transduction can increase the efficiency of M.tb genetic manipulation, this technique is laborious and time consuming (Choudhary et al, 2016; Tufariello et al, 2014; Van Kessel and Hatfull, 2007). Recently a method called ORBIT (oligonucleotide-mediated recombineering followed by Bxb1 integrase targeting) was developed for gene editing in M.tb based on the integration of a targeting oligonucleotide in the genome via homologous recombination mediated by the phage Che9c RecT annealase.…”

Section: Introductionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

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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“…Essential genes are attractive novel drug targets, and the demonstration of essentiality can require modulating the expression of the putative essential gene in a merodiploid strain, an approach that is time consuming, costly, and inefficient in organisms such as mycobacteria that are particularly challenging to genetically modify due to low frequencies of recombination and slow growth (Choudhary et al, 2016). The CRISPRi system overcomes a number of these challenges.…”

Section: Introductionmentioning

confidence: 99%

Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits

et al. 2021

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A greater understanding of the genes involved in antibiotic resistance in Mycobacterium tuberculosis (Mtb) is necessary for the design of improved therapies. Clustered regularly interspaced short palindromic repeat interference (CRISPRi) has been previously utilized in mycobacteria to identify novel drug targets by the demonstration of gene essentiality. The work presented here shows that it can also be usefully applied to the study of non-essential genes involved in antibiotic resistance. The expression of an ADP-ribosyltransferase (Arr) involved in rifampicin resistance in Mycobacterium smegmatis was silenced using CRISPRi and the impact on rifampicin susceptibility was measured. Gene silencing resulted in a decrease in the minimum inhibitory concentration (MIC) similar to that previously reported in an arr deletion mutant. There is contradictory evidence for the toxicity of Streptococcus pyogenes dCas9 (dCas9Spy) in the literature. In this study the expression of dCas9Spy in M. smegmatis showed no impact on viability. Silencing was achieved with concentrations of the aTc inducer lower than previously described and with shorter induction times. Finally, designing small guide RNAs (sgRNAs) that target transcription initiation, or the early stages of elongation had the most impact on rifampicin susceptibility. This study demonstrates that CRISPRi based gene silencing can be as impactful as gene deletion for the study of non-essential genes and further contributes to the knowledge on the design and induction of sgRNAs for CRISPRi. This approach can be applied to other non-essential antimicrobial resistance genes such as drug efflux pumps.

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“…25.170696 doi: bioRxiv preprint vaccine development (3)(4)(5)(6)(7)(8)(9). Various gene editing methods allow researchers to explore genes of interest for their roles in mycobacterial virulence and pathogenicity (10).…”

Section: Introductionmentioning

confidence: 99%

Post-translational knockdown and post-secretional modification of EsxA unambiguously determine the role of EsxA membrane permeabilizing activity in mycobacterial virulence

Bao

Wang

Sun

2020

Preprint

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9Current genetic studies (e.g. gene knockout) have suggested that EsxA and EsxB function as secreted 10 virulence factors that are essential for Mycobaterium tuberculosis (Mtb) virulence, specifically in mediating 11 phagosome rupture and translocation of Mtb to the cytosol of host cells, which further facilitates Mtb 12 intracellular replicating and cell-to-cell spreading. The EsxA-mediated virulence is presumably achieved by 13 its pH-dependent membrane-permeabilizing activity (MPA). However, the data from recent studies have 14 generated a discrepancy regarding to the role of EsxA MPA in mycobacterial virulence with a major concern 15 that genetic manipulations, such as deletion of esxB-esxA operon, may stimulate genetic compensation to 16 produce artifacts and/or affect other co-dependently secreted factors that could be directly involved cytosolic 17 translocation. To avoid the drawbacks of gene knockout, we first engineered a Mycobacterium marinum 18 (Mm) strain, in which a DAS4+ tag was fused to the C-terminus of EsxB to allow inducible knockdown of 19 EsxB (also EsxA) at the post-translational level. We also engineered a Mm strain by fusing a SpyTag to the 20 C-terminus of EsxA, which allows inhibition of EsxA-ST MPA at the post-secretional level through a 21 covalent linkage to SpyCatcher-GFP. Both post-translational knockdown and post-secretional inhibition of 22 EsxA resulted in attenuation of Mm intracellular survival and virulence in macrophages and lung epithelial 23 cells, which unambiguously confirms the role of EsxA MPA in mycobacterial virulence.24 2Genetic studies, such as loss of function by gene deletion and disruption, have suggested that EsxA is a 28 virulence factor essential for mycobacterial virulence. However, its role is questioned because knockout of 29 esxA gene may affect the function or secretion of other related genes. Here, we employed two methods other 30 than gene deletion and disruption to determine EsxA role in mycobacterial virulence. First, we added a 31 degradation signal peptide DAS4+ tag to the C-terminus of EsxB, the chaperon of EsxA so that EsxB-32 DAS4+ could be degraded by protease ClpXP, whose function can be induced by an inducer, ATC. By this 33 way, we were able to control the amount of EsxB and EsxA at the post-translational level. The results 34 showed that ATC inhibited mycobacterial intracellular survival through down-regulating EsxA and EsxB. 35Second method is to take advantage of SpyTag(ST) and SpyCatcher(SC) system. Like DAS4+, ST was 36 fused to C-terminus of EsxA without affecting its expression, secretion and MPA. After secretion, EsxA-ST 37 can be specifically recognized by SC-GFP and form a covalent bond between ST and SC, which blocks the 38 MPA, an activity that directly related to mycobacterial virulence. Endogenous expression of SC-GFP in the 39 infected cells inhibited mycobacterial intracellular survival. In summary, our results demonstrate that 40 knockdown of EsxA at the post-translational level or inhibition of EsxA MPA at the post-secretion...

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Strategies of genome editing in mycobacteria: Achievements and challenges

Cited by 17 publications

References 61 publications

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

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

Re-sensitization of Mycobacterium smegmatis to Rifampicin Using CRISPR Interference Demonstrates Its Utility for the Study of Non-essential Drug Resistance Traits

Post-translational knockdown and post-secretional modification of EsxA unambiguously determine the role of EsxA membrane permeabilizing activity in mycobacterial virulence

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