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 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 A Universal, Genomewide GuideFinder for CRISPR/Cas9 Targeting in Microbial Genomes

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Michelle Spoto ◽  
Changhui Guan ◽  
Elizabeth Fleming ◽  
Julia Oh
Keyword(s):  
Gene Function ◽  
Large Scale ◽  
Essential Gene ◽  
Bacterial Species ◽  
Bacterial Genome ◽  
Model Organisms ◽  
Design Parameters ◽  
Bacterial Genomes ◽  
Wide Range ◽  
User Friendly

ABSTRACT The CRISPR/Cas system has significant potential to facilitate gene editing in a variety of bacterial species. CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) represent modifications of the CRISPR/Cas9 system utilizing a catalytically inactive Cas9 protein for transcription repression and activation, respectively. While CRISPRi and CRISPRa have tremendous potential to systematically investigate gene function in bacteria, few programs are specifically tailored to identify guides in draft bacterial genomes genomewide. Furthermore, few programs offer open-source code with flexible design parameters for bacterial targeting. To address these limitations, we created GuideFinder, a customizable, user-friendly program that can design guides for any annotated bacterial genome. GuideFinder designs guides from NGG protospacer-adjacent motif (PAM) sites for any number of genes by the use of an annotated genome and FASTA file input by the user. Guides are filtered according to user-defined design parameters and removed if they contain any off-target matches. Iteration with lowered parameter thresholds allows the program to design guides for genes that did not produce guides with the more stringent parameters, one of several features unique to GuideFinder. GuideFinder can also identify paired guides for targeting multiplicity, whose validity we tested experimentally. GuideFinder has been tested on a variety of diverse bacterial genomes, finding guides for 95% of genes on average. Moreover, guides designed by the program are functionally useful—focusing on CRISPRi as a potential application—as demonstrated by essential gene knockdown in two staphylococcal species. Through the large-scale generation of guides, this open-access software will improve accessibility to CRISPR/Cas studies of a variety of bacterial species. IMPORTANCE With the explosion in our understanding of human and environmental microbial diversity, corresponding efforts to understand gene function in these organisms are strongly needed. CRISPR/Cas9 technology has revolutionized interrogation of gene function in a wide variety of model organisms. Efficient CRISPR guide design is required for systematic gene targeting. However, existing tools are not adapted for the broad needs of microbial targeting, which include extraordinary species and subspecies genetic diversity, the overwhelming majority of which is characterized by draft genomes. In addition, flexibility in guide design parameters is important to consider the wide range of factors that can affect guide efficacy, many of which can be species and strain specific. We designed GuideFinder, a customizable, user-friendly program that addresses the limitations of existing software and that can design guides for any annotated bacterial genome with numerous features that facilitate guide design in a wide variety of microorganisms.

A novel strategy for identifying mutations that sensitize Drosophila eye development to caffeine and hydroxyurea

Genome ◽  
2006 ◽  
Vol 49 (11) ◽  
pp. 1416-1427 ◽  
Author(s):  
E.A. Silva ◽  
B.J. Lee ◽  
L.S. Caceres ◽  
D. Renouf ◽  
B.R. Vilay ◽  
...  
Keyword(s):  
Gene Function ◽  
Imaginal Disc ◽  
Essential Gene ◽  
Cellular Responses ◽  
Multiple Alleles ◽  
Gene Mutant ◽  
Nonessential Gene ◽  
Disc Cells ◽  
Complementation Groups ◽  
Novel Strategy

This report describes a novel strategy for isolating Drosophila mutants with conditional eye phenotypes that should be generally applicable for identifying genes required for cellular responses to specific drugs. To test the strategy, we screened 3 of the 5 major chromosome arms for hydroxyurea- and (or) caffeine-sensitive (huc) mutants, and isolated mutations affecting 5 different complementation groups. Most of these were represented by single alleles; however, we also isolated multiple alleles of huc29DE gene, an essential gene that is also associated with a nonconditional pupal lethal phenotype. We also identified huc95E mutants, which are extremely sensitive to caffeine. Although huc95E is a nonessential gene, mutant imaginal disc cells undergo caffeine-dependent apoptosis, and huc95E gene function is required for the viability of the organism when mutant larvae are exposed to levels of caffeine that controls can easily tolerate. We have mapped the cytological positions of huc29D and huc95E as a first step toward molecularly characterizing the relevant genes.

scholarly journals Inducible Antisense RNA Expression in the Characterization of Gene Functions in Streptococcus mutans

2005 ◽  
Vol 73 (6) ◽  
pp. 3568-3576 ◽  
Author(s):  
Bing Wang ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Gene Function ◽  
Antisense Rna ◽  
Coenzyme A ◽  
Essential Gene ◽  
Essential Genes ◽  
Expression Vectors ◽  
Rna Expression ◽  
Gene Encoding ◽  
Antisense Dna

ABSTRACT In order to examine gene function in Streptococcus mutans, we have recently initiated an antisense RNA strategy. Toward this end, we have now constructed and evaluated three Escherichia coli-S. mutans shuttle expression vectors with the fruA and scrB promoters from S. mutans, as well as the tetR-controlled tetO promoter from Staphylococcus aureus. Among these, the tetO/tetR system proved to be the most tightly controlled promoter. By using this shuttle plasmid system, modulation of gene function by inducible antisense RNA expression was demonstrated for comC antisense fragments of different sizes as well as for distinct gtfB antisense fragments. It was demonstrated that the size, but not the relative position, of an antisense DNA fragment is important in mediating the antisense phenomenon. Furthermore, by constructing and screening random DNA libraries with the tet expression shuttle system, 78 growth-retarded transformants harboring antisense DNA fragments were also identified. Almost all of them corresponded to homologous essential genes in other bacteria. In addition, a novel essential gene, the coaE gene, encoding dephospho-coenzyme A kinase, which is involved in the final step of coenzyme A catabolism in S. mutans, was identified and characterized. These results suggest that the antisense RNA strategy can be useful for identifying novel essential genes in S. mutans bacteria as well as further characterizing the physiology (including potential virulence factors) of these organisms.

scholarly journals 2-Locus Cleave and Rescue selfish elements harness a recombination rate-dependent generational clock for self limiting gene drive

2020 ◽  
Author(s):  
Georg Oberhofer ◽  
Tobin Ivy ◽  
Bruce A Hay
Keyword(s):  
Dna Sequence ◽  
Gene Function ◽  
Recombination Rate ◽  
High Frequency ◽  
Essential Gene ◽  
Dependent Manner ◽  
Gene Drive ◽  
Rate Dependent

AbstractSelf-limiting gene drive allows control over the spread and fate of linked traits. Cleave and Rescue (ClvR) elements create self-sustaining drive and comprise a DNA sequence-modifying enzyme (Cas9-gRNAs, Cleaver) that disrupts an essential gene, and a tightly linked, uncleavable version of the essential gene (Rescue). ClvR spreads by creating conditions in which those without it die because they lack essential gene function. We show that when ClvR is implemented in a 2-locus format, with key elements – Rescue (and Cargo), and Cas9 and/or gRNAs – located at different genomic positions, spread of the Rescue is self-limiting. Drive strength and duration are determined by a recombination rate-dependent generational clock, providing an important point of control for different ecological and regulatory contexts. We implement 2-locus ClvR in Drosophila. Rescue spreads to high frequency in a Cas9-dependent manner, while the frequency of Cas9 decreases, demonstrating transient drive and loss of future drive potential.

scholarly journals A CRISPR interference platform for selective downregulation of gene expression in Borrelia burgdorferi.

2020 ◽  
Author(s):  
Constantin N. Takacs ◽  
Molly Scott ◽  
Yunjie Chang ◽  
Zachary A. Kloos ◽  
Irnov Irnov ◽  
...  
Keyword(s):  
Gene Expression ◽  
Antibiotic Resistance ◽  
Borrelia Burgdorferi ◽  
Gene Function ◽  
Electron Tomography ◽  
Ease Of Use ◽  
Fast Method ◽  
Cellular Motility ◽  
Crispr Interference ◽  
Resistance Markers

The spirochete Borrelia burgdorferi causes Lyme disease, an increasingly prevalent infection. While previous studies have provided important insight into B. burgdorferi biology, many aspects, including basic cellular processes, remain underexplored. To help speed up the discovery process, we adapted a CRISPR interference (CRISPRi) platform for use in B. burgdorferi. For efficiency and flexibility of use, we generated various CRISPRi template constructs that produce different basal and induced levels of dcas9 and carry different antibiotic resistance markers. We characterized the effectiveness of our CRISPRi platform by targeting the motility and cell morphogenesis genes flaB, mreB, rodA, and ftsI, whose native expression levels span two orders of magnitude. For all four genes, we obtained gene repression efficiencies of at least 95%. We showed by darkfield microscopy and cryo-electron tomography that flagellin (FlaB) depletion reduced the length and number of periplasmic flagella, which impaired cellular motility and resulted in cell straightening. Depletion of FtsI caused cell filamentation, implicating this protein in cell division in B. burgdorferi. Finally, localized cell bulging in MreB- and RodA-depleted cells matched the locations of new peptidoglycan insertion specific to spirochetes of the Borrelia genus. These results therefore implicate MreB and RodA in the particular mode of cell wall elongation of these bacteria. Collectively, our results demonstrate the efficiency and ease of use of our B. burgdorferi CRISPRi platform, which should facilitate future genetic studies of this important pathogen. IMPORTANCE Gene function studies are facilitated by the availability of rapid and easy-to-use genetic tools. Homologous recombination-based methods traditionally used to genetically investigate gene function remain cumbersome to perform in B. burgdorferi, as they often are relatively inefficient. In comparison, our CRISPRi platform offers an easy and fast method to implement as it only requires a single plasmid transformation step and IPTG addition to obtain potent (>95%) downregulation of gene expression. To facilitate studies of various genes in wild-type and genetically modified strains, we provide over 30 CRISPRi plasmids that produce distinct levels of dcas9 expression and carry different antibiotic resistance markers. Our CRISPRi platform represents a useful and efficient complement to traditional genetic and chemical methods to study gene function in B. burgdorferi.

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