Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Rock, Jeremy M.; Hopkins, Forrest; Chavez, Alejandro; Diallo, Marieme; Chase, Michael R.; Gerrick, Elias R; Pritchard, Justin R.; Church, George M.; Rubín, Eric J.; Sassetti, Christopher M.; Schnappinger, Dirk; Fortune, Sarah M.

doi:10.1038/nmicrobiol.2016.274

Cited by 391 publications

(589 citation statements)

References 54 publications

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“…In an attempt to demonstrate inhibition of M. tuberculosis VKOR we have used a CRISPR‐dCas9 system to generate knockdown strains of the essential gene vkor (Rock et al, ). Similar to previous studies, we observed that the silencing of VKOR affects the growth of M. tuberculosis (Sassetti and Rubin, ; Dutton et al, ).…”

Section: Discussionmentioning

confidence: 99%

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

Landeta

McPartland

Tran

et al. 2019

Molecular Microbiology

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Summary In bacteria, disulfide bonds confer stability on many proteins exported to the cell envelope or beyond, including bacterial virulence factors. Thus, proteins involved in disulfide bond formation represent good targets for the development of inhibitors that can act as antibiotics or anti‐virulence agents, resulting in the simultaneous inactivation of several types of virulence factors. Here, we present evidence that the disulfide bond forming enzymes, DsbB and VKOR, are required for Pseudomonas aeruginosa pathogenicity and Mycobacterium tuberculosis survival respectively. We also report the results of a HTS of 216,767 compounds tested against P. aeruginosa DsbB1 and M. tuberculosis VKOR using Escherichia coli cells. Since both P. aeruginosa DsbB1 and M. tuberculosis VKOR complement an E. coli dsbB knockout, we screened simultaneously for inhibitors of each complemented E. coli strain expressing a disulfide‐bond sensitive β‐galactosidase reported previously. The properties of several inhibitors obtained from these screens suggest they are a starting point for chemical modifications with potential for future antibacterial development.

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Section: Discussionmentioning

confidence: 99%

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

Landeta

McPartland

Tran

et al. 2019

Molecular Microbiology

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“…We have recently applied CRISPRi technology to systematically query the importance of ~13,000 genomic features of E. coli in different conditions (Rishi et al 2020 submitted). CRISPRi has been applied to different organisms to study essential genes [68][69][70][71][72][73][74][75][76], and has been recently applied to E. coli to uncover host factors involved in T4, 186 and λ phage infection [51]. Lastly, Dub-Seq uses shotgun cloning of randomly sheared DNA fragments of a host genome on a dual-barcoded replicable plasmid and next-generation sequencing to map the barcodes to the cloned genomic regions.…”

Section: Introductionmentioning

confidence: 99%

High-throughput mapping of the phage resistance landscape inE. coli

Mutalik¹,

Adler²,

Rishi³

et al. 2020

Preprint

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Bacteriophages (phages) are critical players in the dynamics and function of microbial communities and drive processes as diverse as global biogeochemical cycles and human health. Phages tend to be predators finely tuned to attack specific hosts, even down to the strain level, which in turn defend themselves using an array of mechanisms. However, to date, efforts to rapidly and comprehensively identify bacterial host factors important in phage infection and resistance have yet to be fully realized. Here, we globally map the host genetic determinants involved in resistance to 14 phylogenetically diverse double-stranded DNA phages using two model Escherichia coli strains (K-12 and BL21) with known sequence divergence to demonstrate strain-specific differences. Using genome-wide loss-of-function and gain-of-function genetic technologies, we are able to confirm previously described phage receptors as well as uncover a number of previously unknown host factors that confer resistance to one or more of these phages. We uncover differences in resistance factors that strongly align with the susceptibility of K-12 and BL21 to specific phage. We also identify both phage specific mechanisms, such as the unexpected role of cyclic-di-GMP in host sensitivity to phage N4, and more generic defenses, such as the overproduction of colanic acid capsular polysaccharide that defends against a wide array of phages. Our results indicate that host responses to phages can occur via diverse cellular mechanisms. Our systematic and highthroughput genetic workflow to characterize phage-host interaction determinants can be extended to diverse bacteria to generate datasets that allow predictive models of how phagemediated selection will shape bacterial phenotype and evolution. The results of this study and future efforts to map the phage resistance landscape will lead to new insights into the coevolution of hosts and their phage, which can ultimately be used to design better phage therapeutic treatments and tools for precision microbiome engineering.3 Introduction:

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“…Therefore, the unveiling of orthogonal Cas9 variants (such as SpCas9, SaCas9, and St1Cas9) may pave a new avenue to E. coli engineering. In particular, catalytically inactive St1Cas9 (St1dCas9) and SaCas9 (SadCas9) have been developed to suppress target gene expression in Mycobacterium tuberculosis (Rock et al, ), E. coli (Esvelt et al, ) and mammalian cells (Y. Gao et al, ). SpdCas9 can also be fused with the ω subunit of RNA polymerase for specific gene activation in E. coli (Bikard et al, ).…”

Section: Discussionmentioning

confidence: 99%

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

Sung

Lin

et al. 2019

Biotech & Bioengineering

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CRISPR utilizing Cas9 from Streptococcus pyogenes (SpCas9) and CRISPR interference (CRISPRi) employing catalytically inactive SpCas9 (SpdCas9) have gained popularity for Escherichia coli engineering. To integrate the SpdCas9‐based CRISPRi module using CRISPR while avoiding mutual interference between SpCas9/SpdCas9 and their cognate single‐guide RNA (sgRNA), this study aimed at exploring an alternative Cas nuclease orthogonal to SpCas9. We compared several Cas9 variants from different microorganisms such as Staphylococcus aureus (SaCas9) and Streptococcus thermophilius CRISPR1 (St1Cas9) as well as Cas12a derived from Francisella novicida (FnCas12a). At the commonly used E. coli model genes LacZ, we found that SaCas9 and St1Cas9 induced DNA cleavage more effectively than FnCas12a. Both St1Cas9 and SaCas9 were orthogonal to SpCas9 and the induced DNA cleavage promoted the integration of heterologous DNA of up to 10 kb, at which size St1Cas9 was superior to SaCas9 in recombination frequency/accuracy. We harnessed the St1Cas9 system to integrate SpdCas9 and sgRNA arrays for constitutive knockdown of three genes, knock‐in pyc and knockout adhE, without compromising the CRISPRi knockdown efficiency. The combination of orthogonal CRISPR/CRISPRi for metabolic engineering enhanced succinate production while inhibiting byproduct formation and may pave a new avenue to E. coli engineering.

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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Cited by 391 publications

References 54 publications

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

Inhibition of Pseudomonas aeruginosa and Mycobacterium tuberculosis disulfide bond forming enzymes

High-throughput mapping of the phage resistance landscape inE. coli

Combining orthogonal CRISPR and CRISPRi systems for genome engineering and metabolic pathway modulation in Escherichia coli

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