CRISPR interference strategies for studies in essential gene function and live cell fluorescent imaging of DNA elements in Escherichia coli

Amarh et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201803020) visualize for the first time the repair of double-strand breaks during DNA replication. As viewed by live-cell fluorescent imaging of Escherichia coli, repair of replication-dependent breaks is extraordinarily rapid and localized within the cell.

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Expanding the Clostridioides difficile Genetics Toolbox

Journal of Bacteriology ◽

10.1128/jb.00089-19 ◽

2019 ◽

Vol 201 (14) ◽

Cited By ~ 3

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.

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Assessment of plant chaperonin-60 gene function in Escherichia coli

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)50095-8 ◽

1992 ◽

Vol 267 (32) ◽

pp. 23333-23336 ◽

Cited By ~ 2

Author(s):

L.P. Cloney ◽

D.R. Bekkaoui ◽

M.G. Wood ◽

S.M. Hemmingsen

Keyword(s):

Escherichia Coli ◽

Gene Function ◽

Chaperonin 60

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The Escherichia coli bcsB gene is a conditional essential gene in the context of functional cellulose synthesis

FEMS Microbiology Letters ◽

10.1093/femsle/fny232 ◽

2018 ◽

Vol 365 (20) ◽

Author(s):

Enrique Martínez-Carranza ◽

Luis Servín-González ◽

Miguel Cocotl-Yañez ◽

Gloria Soberón-Chávez

Keyword(s):

Escherichia Coli ◽

Essential Gene ◽

Cellulose Synthesis

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The murI gene of Escherichia coli is an essential gene that encodes a glutamate racemase activity.

Journal of Bacteriology ◽

10.1128/jb.175.10.2970-2979.1993 ◽

1993 ◽

Vol 175 (10) ◽

pp. 2970-2979 ◽

Cited By ~ 58

Author(s):

P Doublet ◽

J van Heijenoort ◽

J P Bohin ◽

D Mengin-Lecreulx

Keyword(s):

Escherichia Coli ◽

Essential Gene ◽

Glutamate Racemase

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

mSphere ◽

10.1128/msphere.00086-20 ◽

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.

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HighVia—A Flexible Live-Cell High-Content Screening Pipeline to Assess Cellular Toxicity

SLAS DISCOVERY Advancing Life Sciences ◽

10.1177/2472555220923979 ◽

2020 ◽

Vol 25 (7) ◽

pp. 801-811 ◽

Cited By ~ 1

Author(s):

Alison Howarth ◽

Martin Schröder ◽

Raquel C. Montenegro ◽

David H. Drewry ◽

Heba Sailem ◽

...

Keyword(s):

Cell Viability ◽

High Throughput ◽

Early Stage ◽

Accurate Determination ◽

Live Cell ◽

Fluorescent Imaging ◽

High Content Screening ◽

Imaging Protocol ◽

Target Discovery ◽

Oncology Research

High-content screening to monitor disease-modifying phenotypes upon small-molecule addition has become an essential component of many drug and target discovery platforms. One of the most common phenotypic approaches, especially in the field of oncology research, is the assessment of cell viability. However, frequently used viability readouts employing metabolic proxy assays based on homogeneous colorimetric/fluorescent reagents are one-dimensional, provide limited information, and can in many cases yield conflicting or difficult-to-interpret results, leading to misinterpretation of data and wasted resources.The resurgence of high-content, phenotypic screening has significantly improved the quality and breadth of cell viability data, which can be obtained at the very earliest stages of drug and target discovery. Here, we describe a relatively inexpensive, high-throughput, high-content, multiparametric, fluorescent imaging protocol using a live-cell method of three fluorescent probes (Hoechst, Yo-Pro-3, and annexin V), that is amenable to the addition of further fluorophores. The protocol enables the accurate description and profiling of multiple cell death mechanisms, including apoptosis and necrosis, as well as accurate determination of compound IC50, and has been validated on a range of high-content imagers and image analysis software. To validate the protocol, we have used a small library of approximately 200 narrow-spectrum kinase inhibitors and clinically approved drugs. This fully developed, easy-to-use pipeline has subsequently been implemented in several academic screening facilities, yielding fast, flexible, and rich cell viability data for a range of early-stage high-throughput drug and target discovery programs.

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Fluorescent imaging of cytoplasmic nucleolin in live cells by a functionalized-engineered aptamer

Chemical Communications ◽

10.1039/d0cc06347a ◽

2020 ◽

Vol 56 (91) ◽

pp. 14171-14174

Author(s):

Junfa Yin ◽

Shaokun Chen ◽

Yuanyuan Song ◽

Hailin Wang

Keyword(s):

Cancer Cells ◽

Live Cell ◽

Fluorescent Imaging ◽

Live Cells ◽

Cell Cytoplasm ◽

Graphene Oxides ◽

Normal Cells

A novel biosensing nanoplatform based on aptamer and polymer-grafted graphene oxides can specifically fluorescently visualize nucleolin in live cell cytoplasm, distinguishing cancer cells from normal cells.

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