scholarly journals Expanding the Clostridioides difficile Genetics Toolbox

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Aimee Shen
Keyword(s):  
Gene Silencing ◽  
Gene Function ◽  
Essential Gene ◽  
Transposon Mutagenesis ◽  
Content Type ◽  
Crispr Interference ◽  
Clostridioides Difficile ◽  
Allelic Replacement

ABSTRACT Clostridioides difficile genetics has rapidly advanced in recent years thanks to the development of tools for allelic replacement and transposon mutagenesis. In this Journal of Bacteriology issue, Müh et al. extend the genetics toolbox by developing a CRISPR interference (CRISPRi) strategy for gene silencing in C. difficile (U. Müh, A. G. Pannullo, D. S. Weiss, and C. D. Ellermeier, 2019, J Bacteriol 201:e00711-18. https://doi.org/10.1128/JB.00711-18). The authors demonstrate the tunability and robustness of their CRISPRi system, highlight its utility in studying essential gene function, and discuss exciting new possibilities for dissecting C. difficile physiology.

scholarly journals Investigating essential gene function inMycobacterium tuberculosisusing an efficient CRISPR interference system

2016 ◽  
Vol 44 (18) ◽  
pp. e143-e143 ◽  
Author(s):  
Atul K. Singh ◽  
Xavier Carette ◽  
Lakshmi-Prasad Potluri ◽  
Jared D. Sharp ◽  
Ranfei Xu ◽  
...  
Keyword(s):  
Gene Function ◽  
Essential Gene ◽  
Crispr Interference ◽  
Interference System

scholarly journals Efficient Editing of Malaria Parasite Genome Using the CRISPR/Cas9 System

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Cui Zhang ◽  
Bo Xiao ◽  
Yuanyuan Jiang ◽  
Yihua Zhao ◽  
Zhenkui Li ◽  
...  
Keyword(s):  
Gene Function ◽  
Malaria Parasite ◽  
Gene Deletion ◽  
Plasmodium Yoelii ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Malaria Parasites ◽  
Content Type ◽  
Strand Breaks ◽  
Allelic Replacement

ABSTRACT Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. IMPORTANCE Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite genome will greatly facilitate our ability to elucidate gene function.

scholarly journals A Xylose-Inducible Expression System and a CRISPR Interference Plasmid for Targeted Knockdown of Gene Expression in Clostridioides difficile

2019 ◽  
Vol 201 (14) ◽  
Author(s):  
Ute Müh ◽  
Anthony G. Pannullo ◽  
David S. Weiss ◽  
Craig D. Ellermeier
Keyword(s):  
Gene Expression ◽  
Cell Division ◽  
Fluorescent Protein ◽  
Expression System ◽  
Developed Countries ◽  
Inducible Expression ◽  
Content Type ◽  
Antibiotic Development ◽  
Crispr Interference ◽  
Clostridioides Difficile

ABSTRACT Here we introduce plasmids for xylose-regulated expression and repression of genes in Clostridioides difficile. The xylose-inducible expression vector allows for ∼100-fold induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell to cell. The gene repression plasmid is a CRISPR interference (CRISPRi) system based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively expressed single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing investigators to control the timing and extent of gene silencing, as demonstrated here by dose-dependent repression of a chromosomal gene for a red fluorescent protein (maximum repression, ∼100-fold). To validate the utility of CRISPRi for deciphering gene function in C. difficile, we knocked down the expression of three genes involved in the biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA, and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected phenotypes associated with the loss of FtsZ and SlpA and revealed that the previously uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell division, and protection against lysis. IMPORTANCE Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in developed countries. A better understanding of the basic biology of this devastating pathogen might lead to novel approaches for preventing or treating C. difficile infections. Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy depletion of target proteins in C. difficile. Besides bypassing the lengthy process of mutant construction, CRISPRi can be used to study the function of essential genes, which are particularly important targets for antibiotic development.

scholarly journals A High-Efficacy CRISPR Interference System for Gene Function Discovery in Zymomonas mobilis

2020 ◽  
Vol 86 (23) ◽  
Author(s):  
Amy B. Banta ◽  
Amy L. Enright ◽  
Cheta Siletti ◽  
Jason M. Peters
Keyword(s):  
Gene Function ◽  
Zymomonas Mobilis ◽  
Strain Engineering ◽  
Essential Genes ◽  
Biofuel Production ◽  
Alcohol Tolerance ◽  
High Efficacy ◽  
Content Type ◽  
Crispr Interference ◽  
Function Discovery

ABSTRACT Zymomonas mobilis is a promising biofuel producer due to its high alcohol tolerance and streamlined metabolism that efficiently converts sugar to ethanol. Z. mobilis genes are poorly characterized relative to those of model bacteria, hampering our ability to rationally engineer the genome with pathways capable of converting sugars from plant hydrolysates into valuable biofuels and bioproducts. Many of the unique properties that make Z. mobilis an attractive biofuel producer are controlled by essential genes; however, these genes cannot be manipulated using traditional genetic approaches (e.g., deletion or transposon insertion) because they are required for viability. CRISPR interference (CRISPRi) is a programmable gene knockdown system that can precisely control the timing and extent of gene repression, thus enabling targeting of essential genes. Here, we establish a stable, high-efficacy CRISPRi system in Z. mobilis that is capable of perturbing all genes—including essential genes. We show that Z. mobilis CRISPRi causes either strong knockdowns (>100-fold) using single guide RNA (sgRNA) spacers that perfectly match target genes or partial knockdowns using spacers with mismatches. We demonstrate the efficacy of Z. mobilis CRISPRi by targeting essential genes that are universally conserved in bacteria, are key to the efficient metabolism of Z. mobilis, or underlie alcohol tolerance. Our Z. mobilis CRISPRi system will enable comprehensive gene function discovery, opening a path to rational design of biofuel production strains with improved yields. IMPORTANCE Biofuels produced by microbial fermentation of plant feedstocks provide renewable and sustainable energy sources that have the potential to mitigate climate change and improve energy security. Engineered strains of the bacterium Z. mobilis can convert sugars extracted from plant feedstocks into next-generation biofuels like isobutanol; however, conversion by these strains remains inefficient due to key gaps in our knowledge about genes involved in metabolism and stress responses such as alcohol tolerance. Here, we develop CRISPRi as a tool to explore gene function in Z. mobilis. We characterize genes that are essential for growth, required to ferment sugar to ethanol, and involved in resistance to isobutanol. Our Z. mobilis CRISPRi system makes it straightforward to define gene function and can be applied to improve strain engineering and increase biofuel yields.

scholarly journals A Conditional Protein Degradation System To Study Essential Gene Function in Cryptosporidium parvum

mBio ◽  
2020 ◽  
Vol 11 (4) ◽  
Author(s):  
Hadi H. Choudhary ◽  
Maria G. Nava ◽  
Brina E. Gartlan ◽  
Savannah Rose ◽  
Sumiti Vinayak
Keyword(s):  
Cryptosporidium Parvum ◽  
Gene Function ◽  
Essential Gene ◽  
Critical Role ◽  
Essential Genes ◽  
Parasite Line ◽  
Asexual Parasite ◽  
Content Type ◽  
Genetic Tool ◽  
Transgenic Parasites

ABSTRACT Cryptosporidium spp., protozoan parasites, are a leading cause of global diarrhea-associated morbidity and mortality in young children and immunocompromised individuals. The limited efficacy of the only available drug and lack of vaccines make it challenging to treat and prevent cryptosporidiosis. Therefore, the identification of essential genes and understanding their biological functions are critical for the development of new therapies. Currently, there is no genetic tool available to investigate the function of essential genes in Cryptosporidium spp. Here, we describe the development of the first conditional system in Cryptosporidium parvum. Our system utilizes the Escherichia coli dihydrofolate reductase degradation domain (DDD) and the stabilizing compound trimethoprim (TMP) for conditional regulation of protein levels in the parasite. We tested our system on the calcium-dependent protein kinase-1 (CDPK1), a leading drug target in C. parvum. By direct knockout strategy, we establish that cdpk1 is refractory to gene deletion, indicating its essentiality for parasite survival. Using CRISPR/Cas9, we generated transgenic parasites expressing CDPK1 with an epitope tag, and localization studies indicate its expression during asexual parasite proliferation. We then genetically engineered C. parvum to express CDPK1 tagged with DDD. We demonstrate that TMP can regulate CDPK1 levels in this stable transgenic parasite line, thus revealing the critical role of this kinase in parasite proliferation. Further, these transgenic parasites show TMP-mediated regulation of CDPK1 levels in vitro and an increased sensitivity to kinase inhibitor upon conditional knockdown. Overall, this study reports the development of a powerful conditional system that can be used to study essential genes in Cryptosporidium. IMPORTANCE Cryptosporidium parvum and Cryptosporidium hominis are leading pathogens responsible for diarrheal disease (cryptosporidiosis) and deaths in infants and children below 5 years of age. There are no effective treatment options and no vaccine for cryptosporidiosis. Therefore, there is an urgent need to identify essential gene targets and uncover their biological function to accelerate the development of new and effective anticryptosporidial drugs. Current genetic tool allows targeted disruption of gene function but leads to parasite lethality if the gene is essential for survival. In this study, we have developed a genetic tool for conditional degradation of proteins in Cryptosporidium spp., thus allowing us to study the function of essential genes. Our conditional system expands the molecular toolbox for Cryptosporidium, and it will help us to understand the biology of this important human diarrheal pathogen for the development of new drugs and vaccines.

scholarly journals A Genetic Resource for Rapid and Comprehensive Phenotype Screening of Nonessential Staphylococcus aureus Genes

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Paul D. Fey ◽  
Jennifer L. Endres ◽  
Vijaya Kumar Yajjala ◽  
Todd J. Widhelm ◽  
Robert J. Boissy ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Large Scale ◽  
Essential Gene ◽  
Protease Production ◽  
Single Mutation ◽  
Mutant Library ◽  
Extracellular Proteases ◽  
Pigment Formation ◽  
Content Type ◽  
Allelic Replacement

ABSTRACTTo enhance the research capabilities of investigators interested inStaphylococcus aureus, the Nebraska Center for Staphylococcal Research (CSR) has generated a sequence-defined transposon mutant library consisting of 1,952 strains, each containing a single mutation within a nonessential gene of the epidemic community-associated methicillin-resistantS. aureus(CA-MRSA) isolate USA300. To demonstrate the utility of this library for large-scale screening of phenotypic alterations, we spotted the library on indicator plates to assess hemolytic potential, protease production, pigmentation, and mannitol utilization. As expected, we identified many genes known to function in these processes, thus validating the utility of this approach. Importantly, we also identified genes not previously associated with these phenotypes. In total, 71 mutants displayed differential hemolysis activities, the majority of which were not previously known to influence hemolysin production. Furthermore, 62 mutants were defective in protease activity, with only 14 previously demonstrated to be involved in the production of extracellular proteases. In addition, 38 mutations affected pigment formation, while only 7 influenced mannitol fermentation, underscoring the sensitivity of this approach to identify rare phenotypes. Finally, 579 open reading frames were not interrupted by a transposon, thus providing potentially new essential gene targets for subsequent antibacterial discovery. Overall, the Nebraska Transposon Mutant Library represents a valuable new resource for the research community that should greatly enhance investigations of this important human pathogen.IMPORTANCEInfections caused byStaphylococcus aureuscause significant morbidity and mortality in both community and hospital environments. Specific-allelic-replacement mutants are required to study the biology of this organism; however, this process is costly and time-consuming. We describe the construction and validation of a sequence-defined transposon mutant library available for use by the scientific community through the Network on Antimicrobial Resistance inStaphylococcus aureus(NARSA) strain repository. In addition, complementary resources, including a website (http://app1.unmc.edu/fgx/) and genetic tools that expedite the allelic replacement of the transposon in the mutants with useful selectable markers and fluorescent reporter fusions, have been generated. Overall, this library and associated tools will have a significant impact on studies investigatingS. aureuspathogenesis and biology and serve as a useful paradigm for the study of other bacterial systems.

scholarly journals Polar Effects of Transposon Insertion into a Minimal Bacterial Genome

2019 ◽  
Vol 201 (19) ◽  
Author(s):  
Clyde A. Hutchison ◽  
Chuck Merryman ◽  
Lijie Sun ◽  
Nacyra Assad-Garcia ◽  
R. Alexander Richter ◽  
...  
Keyword(s):  
Gene Function ◽  
Essential Gene ◽  
Global Analysis ◽  
Bacterial Genome ◽  
Transposon Mutagenesis ◽  
Protein Coding ◽  
Transposon Insertion ◽  
Nonessential Gene ◽  
Intergenic Regions

ABSTRACT Global transposon mutagenesis is a valuable tool for identifying genes required for cell viability. Here we present a global analysis of the orientation of viable Tn5-Puror (Tn5-puromycin resistance) insertions into the near-minimal bacterial genome of JCVI-syn2.0. Sixteen of the 478 protein-coding genes show a noticeable asymmetry in the orientation of disrupting insertions of Tn5-Puror. Ten of these are located in operons, upstream of essential or quasi-essential genes. Inserts transcribed in the same direction as the downstream gene are favored, permitting read-through transcription of the essential or quasi-essential gene. Some of these genes were classified as quasi-essential solely because of polar effects on the expression of downstream genes. Three genes showing asymmetry in Tn5-Puror insertion orientation prefer the orientation that avoids collisions between read-through transcription of Tn5-Puror and transcription of an adjacent gene. One gene (JCVISYN2_0132 [abbreviated here as “_0132”]) shows a strong preference for Tn5-Puror insertions transcribed upstream, away from the downstream nonessential gene _0133. This suggested that expression of _0133 due to read-through from Tn5-Puror is lethal when _0132 function is disrupted by transposon insertion. This led to the identification of genes _0133 and _0132 as a toxin-antitoxin pair. The three remaining genes show read-through transcription of Tn5-Puror directed downstream and away from sizable upstream intergenic regions (199 bp to 363 bp), for unknown reasons. In summary, polar effects of transposon insertion can, in a few cases, affect the classification of genes as essential, quasi-essential, or nonessential and sometimes can give clues to gene function. IMPORTANCE In studies of the minimal genetic requirements for life, we used global transposon mutagenesis to identify genes needed for a minimal bacterial genome. Transposon insertion can disrupt the function of a gene but can also have polar effects on the expression of adjacent genes. In the Tn5-Puror construct used in our studies, read-through transcription from Tn5-Puror can drive expression of downstream genes. This results in a preference for Tn5-Puror insertions transcribed toward a downstream essential or quasi-essential gene within the same operon. Such polar effects can have an impact on the classification of genes as essential, quasi-essential, or nonessential, but this has been observed in only a few cases. Also, polar effects of Tn5-Puror insertion can sometimes give clues to gene function.

scholarly journals Bacterial Resistance to Antisense Peptide Phosphorodiamidate Morpholino Oligomers

2012 ◽  
Vol 56 (12) ◽  
pp. 6147-6153 ◽  
Author(s):  
Susan E. Puckett ◽  
Kaleb A. Reese ◽  
Georgi M. Mitev ◽  
Valerie Mullen ◽  
Rudd C. Johnson ◽  
...  
Keyword(s):  
Bacterial Resistance ◽  
Acyl Carrier Protein ◽  
Essential Gene ◽  
Resistant Mutant ◽  
Resistant Isolate ◽  
Carrier Protein ◽  
Bacterial Gene ◽  
Content Type ◽  
Bacterial Gene Expression ◽  
Phosphorodiamidate Morpholino Oligomers

ABSTRACTPeptide phosphorodiamidate morpholino oligomers (PPMOs) are synthetic DNA mimics that bind cRNA and inhibit bacterial gene expression. The PPMO (RFF)3RXB-AcpP (where R is arginine, F, phenylalanine, X is 6-aminohexanoic acid, B is β-alanine, and AcpP is acyl carrier protein) is complementary to 11 bases of the essential geneacpP(which encodes acyl carrier protein). The MIC of (RFF)3RXB-AcpP was 2.5 μM (14 μg/ml) inEscherichia coliW3110. The rate of spontaneous resistance ofE. colito (RFF)3RXB-AcpP was 4 × 10−7mutations/cell division. A spontaneous (RFF)3RXB-AcpP-resistant mutant (PR200.1) was isolated. The MIC of (RFF)3RXB-AcpP was 40 μM (224 μg/ml) for PR200.1. The MICs of standard antibiotics for PR200.1 and W3110 were identical. The sequence ofacpPwas identical in PR200.1 and W3110. PR200.1 was also resistant to other PPMOs conjugated to (RFF)3RXB or peptides with a similar composition or pattern of cationic and nonpolar residues. Genomic sequencing of PR200.1 identified a mutation insbmA, which encodes an active transport protein. In separate experiments, a (RFF)3RXB-AcpP-resistant isolate (RR3) was selected from a transposome library, and the insertion was mapped tosbmA. Genetic complementation of PR200.1 or RR3 withsbmArestored susceptibility to (RFF)3RXB-AcpP. Deletion ofsbmAcaused resistance to (RFF)3RXB-AcpP. We conclude that resistance to (RFF)3RXB-AcpP was linked to the peptide and not the phosphorodiamidate morpholino oligomer, dependent on the composition or repeating pattern of amino acids, and caused by mutations insbmA. The data further suggest that (RFF)3R-XB PPMOs may be transported across the plasma membrane by SbmA.

scholarly journals A cassava common mosaic virus vector for virus-induced gene silencing in cassava

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Decai Tuo ◽  
Peng Zhou ◽  
Pu Yan ◽  
Hongguang Cui ◽  
Yang Liu ◽  
...  
Keyword(s):  
Gene Silencing ◽  
Mosaic Virus ◽  
Gene Function ◽  
Viral Vector ◽  
Function Analysis ◽  
Systemic Infection ◽  
Cdna Clones ◽  
Virus Induced Gene Silencing ◽  
Efficient Gene ◽  
Gene Function Analysis

Abstract Background Cassava is an important crop for food security and industry in the least-developed and developing countries. The completion of the cassava genome sequence and identification of large numbers of candidate genes by next-generation sequencing provide extensive resources for cassava molecular breeding and increase the need for rapid and efficient gene function analysis systems in cassava. Several plant virus-induced gene silencing (VIGS) systems have been developed as reverse genetic tools for rapid gene function analysis in cassava. However, these VIGS vectors could cause severe viral symptoms or inefficient gene silencing. Results In this study, we constructed agroinfection-compatible infectious cDNA clones of cassava common mosaic virus isolate CM (CsCMV-CM, genus Potexvirus, family Alphaflexiviridae) that causes systemic infection with mild symptoms in cassava. CsCMV-CM was then modified to a viral vector carrying the Nimble cloning frame, which facilitates the rapid and high-throughput cloning of silencing fragments into the viral genome. The CsCMV-based vector successfully silenced phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) in different cassava varieties and Nicotiana benthamiana. The silencing of the ChlI gene could persist for more than two months. Conclusions This CsCMV-based VIGS system provides a new tool for rapid and efficient gene function studies in cassava.

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