Pseudomonas aeruginosa: Methods and Protocols (Methods in Molecular Biology, 2721) 1071634720, 9781071634721

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Methods in Molecular Biology 2721

Giovanni Bertoni · Silvia Ferrara  Editors

Pseudomonas aeruginosa Methods and Protocols

METHODS

IN

MOLECULAR BIOLOGY

Series Editor John M. Walker School of Life and Medical Sciences University of Hertfordshire Hatfield, Hertfordshire, UK

For further volumes: http://www.springer.com/series/7651

For over 35 years, biological scientists have come to rely on the research protocols and methodologies in the critically acclaimed Methods in Molecular Biology series. The series was the first to introduce the step-by-step protocols approach that has become the standard in all biomedical protocol publishing. Each protocol is provided in readily-reproducible step-by step fashion, opening with an introductory overview, a list of the materials and reagents needed to complete the experiment, and followed by a detailed procedure that is supported with a helpful notes section offering tips and tricks of the trade as well as troubleshooting advice. These hallmark features were introduced by series editor Dr. John Walker and constitute the key ingredient in each and every volume of the Methods in Molecular Biology series. Tested and trusted, comprehensive and reliable, all protocols from the series are indexed in PubMed.

Pseudomonas aeruginosa Methods and Protocols

Edited by

Giovanni Bertoni and Silvia Ferrara Department of Biosciences, Università degli Studi di Milano, Milan, Milano, Italy

Editors Giovanni Bertoni Department of Biosciences Universita` degli Studi di Milano Milan, Milano, Italy

Silvia Ferrara Department of Biosciences Universita` degli Studi di Milano Milan, Milano, Italy

ISSN 1064-3745 ISSN 1940-6029 (electronic) Methods in Molecular Biology ISBN 978-1-0716-3472-1 ISBN 978-1-0716-3473-8 (eBook) https://doi.org/10.1007/978-1-0716-3473-8 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Humana imprint is published by the registered company Springer Science+Business Media, LLC, part of Springer Nature. The registered company address is: 1 New York Plaza, New York, NY 10004, U.S.A. Paper in this product is recyclable.

Preface Ubiquitous and versatile are the adjectives most frequently used to define the bacterium Pseudomonas aeruginosa. There are several questions that we can ask when trying to understand how P. aeruginosa was able to evolve the ability to exploit matter and energy sources in many different and often harsh environments. The answers to many of these questions are provided by research focused on understanding the molecular mechanisms that allow P. aeruginosa to behave as an opportunistic pathogen in humans. From these studies, we have learned that P. aeruginosa has great metabolic, regulatory, and genetic adaptability. However, P. aeruginosa is a complex and many-sided organism, and the integration of the puzzle pieces into a single figure is extremely difficult. In addition, much of the work to date has involved populations of P. aeruginosa cells that do not interact with the host, and extrapolating a host-oriented conclusion from these results is not always straightforward. Indeed, there is increasing evidence of the impact of the host environment on the dynamics of P. aeruginosa infection, including heterogeneity in the adaptive response within the infecting population. Therefore, it is more and more necessary that we seek to reproduce the crosstalk between P. aeruginosa and the human host and learn to account for this heterogeneity if we are to use the right pieces to reconstruct the puzzle. Emerging techniques of transcriptomics at the single bacterial cell level and the development of organoid-based infection models of the human airways with monitoring of biofilm dynamics over time and dual (both pathogen and host) transcriptomic analysis are moving in this direction. We are aware that this is a major challenge for the future, to be addressed both with the methods described in the previous mighty edition of Pseudomonas, edited by Alain Filloux and Juan-Luis Ramos in 2014, and with protocols that have been refined over the last decade and that we have collected in this book. Milan, Milano, Italy

Giovanni Bertoni Silvia Ferrara

v

Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

CRISPR-BASED GENOME EDITING

1 CRISPR/Cas9-based Genome Editing of Pseudomonas aeruginosa. . . . . . . . . . . . Weizhong Chen and Quanjiang Ji 2 Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Michelle A. Yu, Amy B. Banta, Ryan D. Ward, Neha K. Prasad, Michael S. Kwon, Oren S. Rosenberg, and Jason M. Peters

PART II

v ix

3

13

GENETIC TOOLS AND BIOSENSORS

3 Engineering Green-light-responsive Heterologous Gene Expression in Pseudomonas . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35 Angeles Hueso-Gil, Bele´n Calles, and Vı´ctor de Lorenzo 4 Fluorescence-based Evaluation of Cyclic di-GMP Levels in Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45 Silvia Santoro, Giovanni Bertoni, and Silvia Ferrara 5 Whole-Cell Biosensors for Qualitative and Quantitative Analysis of Quorum Sensing Signal Molecules and the Investigation of Quorum Quenching Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55 Marta Mellini, Morgana Letizia, Livia Leoni, and Giordano Rampioni 6 A Pseudomonas aeruginosa-Suitable Fluorescent Reporter System for Analyzing Small RNA-Mediated Regulation of Target mRNAs . . . . . . . . . . . . 69 Silvia Santoro, Giovanni Bertoni, and Silvia Ferrara 7 The Pseudomonas aeruginosa Resistome: Permanent and Transient Antibiotic Resistance, an Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85 Fernando Sanz-Garcı´a, Pablo Laborda, Luz Edith Ochoa-Sa´nchez, Jose´ Luis Martı´nez, and Sara Hernando-Amado 8 Biosensors for Inducers of Transient Antibiotic Resistance . . . . . . . . . . . . . . . . . . . 103 Pablo Laborda, Manuel Alcalde-Rico, Teresa Gil-Gil, Jose´ Luis Martı´nez, and Paula Blanco

PART III

SECRETED FACTORS AND MICROSCALE ANALYSIS OF BIOFILM

9 Pseudomonas aeruginosa Soluble Pyocins as Antibacterial Weapons . . . . . . . . . . . . 125 Pierre Cornelis, Jozef Dingemans, and Christine Baysse 10 Assays for Studying Pseudomonas aeruginosa Secreted Proteases . . . . . . . . . . . . . . 137 Alessandra Fortuna, Diletta Collalto, Giordano Rampioni, and Livia Leoni

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viii

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Contents

Single Microcolony Diffusion Analysis in Pseudomonas aeruginosa Biofilms . . . . 153 Jagadish Sankaran, Scott A. Rice, and Thorsten Wohland

PART IV

GENOME-SCALE APPROACHES

12

Broad Genome Sequencing of Environmental and Clinical Strains and Genotyping . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 Alfonso Esposito and Silvano Piazza 13 Genome-Scale Analysis of the Structure and Function of RNA Pathways and Networks in Pseudomonas aeruginosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183 Silvia Ferrara and Giovanni Bertoni 14 In-Depth Quantitative Proteomics Analysis of the Pseudomonas aeruginosa Secretome . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Dimitrios Lampaki, Andreas Diepold, and Timo Glatter

PART V 15

16

HOST-PATHOGEN INTERACTION

Improving the Predictive Value of Preclinical Mouse Models of Pseudomonas aeruginosa Respiratory Infection to Evaluate Antibiotic Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Cristina Cigana, Alice Rossi, Beatriz Alcala´-Franco, and Alessandra Bragonzi Emerging In Vitro Models for the Study of Infection and Pathogenesis of Pseudomonas aeruginosa and Testing of Antibacterial Agents. . . . . . . . . . . . . . . 233 Tarcisio Brignoli, Silvia Ferrara, and Giovanni Bertoni

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

Contributors BEATRIZ ALCALA´-FRANCO • Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Disease, IRCSS San Raffaele Scientific Institute, Milan, Italy MANUEL ALCALDE-RICO • Grupo de Resistencia Antimicrobiana en Bacterias Patogenas y Ambientales (GRABPA), Instituto de Biologı´a, Facultad de Ciencias, Pontificia Universidad Catolica de Valparaı´so, Valparaı´so, Chile; Millennium Nucleus for Collaborative Research on Bacterial Resistance (MICROB-R), Valparaı´so, Chile AMY B. BANTA • Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA; Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA CHRISTINE BAYSSE • Institut de Ge´ne´tique et de De´veloppement de Rennes (IGDR), CNRS UMR 6290, Universite´ de Rennes, Rennes, France ` degli Studi di Milano, Milan, GIOVANNI BERTONI • Department of Biosciences, Universita Italy PAULA BLANCO • Molecular Basis of Adaptation, Departamento de Sanidad Animal, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain ALESSANDRA BRAGONZI • Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Disease, IRCSS San Raffaele Scientific Institute, Milan, Italy ` degli Studi di Milano, Milan, TARCISIO BRIGNOLI • Department of Biosciences, Universita Italy BELE´N CALLES • Systems Biology Department, Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain WEIZHONG CHEN • School of Physical Science and Technology, ShanghaiTech University, Shanghai, China CRISTINA CIGANA • Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Disease, IRCSS San Raffaele Scientific Institute, Milan, Italy DILETTA COLLALTO • Department of Science, University Roma Tre, Rome, Italy PIERRE CORNELIS • Vrije Universiteit Brussel, Microbiology Group, Brussels, Belgium VI´CTOR DE LORENZO • Systems Biology Department, Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain ANDREAS DIEPOLD • Max Planck Institute for Terrestrial Microbiology, Marburg, Germany JOZEF DINGEMANS • Vrije Universiteit Brussel, Microbiology Group, Brussels, Belgium ALFONSO ESPOSITO • Faculty of Medicine and Surgery, “Kore” University of Enna, Enna, Italy ` degli Studi di Milano, Milan, SILVIA FERRARA • Department of Biosciences, Universita Milano, Italy ALESSANDRA FORTUNA • Department of Science, University Roma Tre, Rome, Italy TERESA GIL-GIL • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain TIMO GLATTER • Max Planck Institute for Terrestrial Microbiology, Marburg, Germany SARA HERNANDO-AMADO • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain

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Contributors

ANGELES HUESO-GIL • Systems Biology Department, Centro Nacional de Biotecnologı´a (CNB-CSIC), Madrid, Spain; Centro de Biotecnologı´a y Genomica de Plantas (UPM-INIA/CSIC), Madrid, Spain QUANJIANG JI • School of Physical Science and Technology, ShanghaiTech University, Shanghai, China MICHAEL S. KWON • Division of Pulmonary, Critical Care, Allergy, and Sleep Medicine, Department of Medicine, University of California, San Francisco, CA, USA PABLO LABORDA • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain DIMITRIOS LAMPAKI • Max Planck Institute for Immunology and Epigenetics, Freiburg, Germany LIVIA LEONI • Department of Science, University Roma Tre, Rome, Italy MORGANA LETIZIA • Department of Science, University Roma Tre, Rome, Italy JOSE´ LUIS MARTI´NEZ • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain MARTA MELLINI • Department of Science, University Roma Tre, Rome, Italy LUZ EDITH OCHOA-SA´NCHEZ • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain JASON M. PETERS • Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA; Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI, USA; Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, USA; Department of Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, WI, USA SILVANO PIAZZA • Computational Biology Unit, International Centre for Genetic Engineering and Biotechnology (ICGEB), Trieste, Italy NEHA K. PRASAD • Chan Zuckerberg Biohub, San Francisco, CA, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA GIORDANO RAMPIONI • Department of Science, University Roma Tre, Rome, Italy; IRCCS Fondazione Santa Lucia, Rome, Italy SCOTT A. RICE • Singapore Centre for Environmental Life Science and Engineering, Singapore, Singapore; University of Technology Sydney, Ultimo, NSW, Australia; CSIRO, Agriculture and Food, Microbiomes for One Systems Health, Coopers Plains, QLD, Australia OREN S. ROSENBERG • Chan Zuckerberg Biohub, San Francisco, CA, USA; Department of Medicine, University of California, San Francisco, San Francisco, CA, USA; Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA ALICE ROSSI • Infections and Cystic Fibrosis Unit, Division of Immunology, Transplantation and Infectious Disease, IRCSS San Raffaele Scientific Institute, Milan, Italy JAGADISH SANKARAN • Genome Institute of Singapore, Singapore, Singapore ` degli Studi di Milano, Milan, Italy SILVIA SANTORO • Department of Biosciences, Universita FERNANDO SANZ-GARCI´A • Centro Nacional de Biotecnologı´a, CSIC, Madrid, Spain RYAN D. WARD • Pharmaceutical Sciences Division, School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA; Laboratory of Genetics, University of WisconsinMadison, Madison, WI, USA THORSTEN WOHLAND • Departments of Biological Sciences and Chemistry, National University of Singapore, Singapore, Singapore MICHELLE A. YU • Division of Pulmonary, Critical Care, Allergy, and Sleep Medicine, Department of Medicine, University of California, San Francisco, CA, USA

Part I CRISPR-Based Genome Editing

Chapter 1 CRISPR/Cas9-based Genome Editing of Pseudomonas aeruginosa Weizhong Chen and Quanjiang Ji Abstract Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has been developed as a robust genome engineering tool in a variety of organisms attributed to its high efficiency and versatility. In this chapter, we described the detailed procedures of CRISPR-Cas9-based genetic manipulation in Pseudomonas aeruginosa, including precise gene deletion and insertion via Cas9-mediated DNA double-strand break and homologous recombination repair. In addition, we provided a detailed protocol for cytidine base editor, a highly efficient gene inactivation and point mutation tool in Pseudomonas aeruginosa. Key words Pseudomonas aeruginosa, CRISPR-Cas9, Genome editing, Base editing, Gene inactivation

1

Introduction Clustered regularly interspaced short palindromic repeats (CRISPR) systems, which were originally discovered in many bacteria and most archaea, have a defensive function against invading DNA [1]. Among these CRISPR systems, the type II CRISPR– Cas9 system from Streptococcus pyogenes (SpCas9) is the best characterized system and has been harnessed for robust genome editing in diverse organisms, such as mammalian cells [2], plants [3], yeast [4], and bacteria [5, 6]. In the CRISPR–Cas9 system, the Cas9 DNA nuclease forms a complex with an artificial chimeric single guide RNA (sgRNA) which directs the Cas9 nuclease to specific genomic locus via complementary base pairing when a protospacer adjacent motif (PAM, e.g., 5′-NGG-3′ for Streptococcus pyogenes Cas9) is present at the 3′ end of the target locus [7, 8]. After binding to the target site, the Cas9 nuclease cleaves the doublestranded DNA and generates a double-strand break (DSB) within the base-pairing region (Fig. 1a). Given that most bacterial cells do not possess the non-homologous end-joining (NHEJ) repair pathway, the cells must undergo homologous recombination to repair

Giovanni Bertoni and Silvia Ferrara (eds.), Pseudomonas aeruginosa: Methods and Protocols, Methods in Molecular Biology, vol. 2721, https://doi.org/10.1007/978-1-0716-3473-8_1, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Weizhong Chen and Quanjiang Ji

Fig. 1 The graphic illustration of the detailed mechanism of CRISPR-Cas9-mediated genome editing method (a) and cytidine base editing method (b) in P. aeruginosa

the DSB of the genome. During the process of homology-directed repair, sequence-specific deletions, insertions, or point mutations can be achieved through the recombination of the target locus with an exogenously supplied DNA donor template (Fig. 1a) [9]. Therefore, it is possible to achieve a one-step seamless genome editing in Pseudomonas aeruginosa with the utilization of the CRISPR-Cas9 system. More recently, deaminase-mediated base-editing systems which catalyze the programmable base conversion in the specific genome sites have been developed, providing a new strategy for genome editing in living cells [10–13]. By engineering the fusion of different deaminases with a Cas9 nickase (D10A) or a dead Cas9 (D10AH840A), several base editors have been developed, including cytidine base editors (CBEs) for C to T conversion [10], adenosine base editors (ABEs) for A to G conversion [11], and glycosylase base editors (GBEs) for C to G conversion [12, 13]. By precisely catalyzing the conversion of the four coding codons (CAA, CAG, CGA, or TGG) into stop codons (TAA, TAG, or TGA), the CBE is capable of inactivating the target genes via introducing a premature stop codon in gene open reading frames (Fig. 1b). Comparing with the CRISPR-Cas9-based genome editing aforementioned, the CBE does not generate DSB. Therefore, this method does not require the donor template or sacrifice transformation CFUs, making it to be a simple and efficient genetic manipulation tool in Pseudomonas aeruginosa, especially in some clinical strains with low transformation efficiency.

CRISPR/Cas9-based Genome Editing

5

In our previous study, we developed a pCasPA/pACRISPR two-plasmid system for efficient and scarless genetic manipulation in P. aeruginosa by harnessing the CRISPR-Cas9 and the phage λRed recombination systems [14]. In addition, by engineering the fusion of the rat cytidine deaminase APOBEC1 and the Cas9 nickase, we further developed a base editing plasmid pnCasPA-BEC, enabling highly efficient gene inactivation and point mutations in P. aeruginosa [14]. This chapter aims to provide a detailed procedure of how to use these two tools in the genome editing of P. aeruginosa.

2

Materials

2.1 Construction of the Editing Plasmids

1. DH5α competent cell. 2. Luria-Bertani (LB) broth. 3. LB agar broth. 4. pCasPA plasmid (Addgene Catalog No. 113347). 5. pACRISPR plasmid (Addgene Catalog No. 113348). 6. pnCasPA-BEC plasmid (Addgene Catalog No. 113349). 7. Tetracycline. 8. Carbenicillin. 9. Gentamycin. 10. BsaI-HF. 11. T4 DNA ligase. 12. XbaI. 13. XhoI. 14. High-fidelity DNA polymerase. 15. PCR product purification kit. 16. Gibson assembly kit. 17. Plasmid mini-prep kit. 18. PCR thermocycler. 19. DNA gel electrophoresis. 20. 0.2 mL PCR tubes. 21. ddH2O.

2.2 Preparation of P. aeruginosa Electrocompetent Cells

1. P. aeruginosa isolates. 2. 10% v/v sterile glycerol. 3. 50 mL conical tubes. 4. 1.5 mL Eppendorf tubes. 5. Liquid nitrogen.

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Weizhong Chen and Quanjiang Ji

2.3 Genome Editing in P. aeruginosa

1. 1 mm electroporation cuvette. 2. Gene Pulser Xcell™ Electroporation System (Bio-Rad). 3. L-arabinose. 4. Sucrose.

3

Methods

3.1 Construction of the Editing Plasmid 3.1.1 Insertion of the Suitable Spacer into the pACRISPR Plasmid

1. Select a suitable 20-nt spacer sequence immediately preceding the 5′-NGG-3′ PAM in the target gene of P. aeruginosa (see Note 1). As shown in Fig. 2a, b, the FWD and REV primers were designed according to the BsaI sites of the pACRISPR plasmid. 2. Anneal the two oligos in a sterile PCR tube as follows: Component

Volume

Final concentration

10 μM FWD primer

10 μL

2 μM

10 μM REV primer

10 μL

2 μM

1 M NaCl

5 μL

100 mM

ddH2O to 50 μL

and incubate the solution at 95 °C for 5 min. Then slowly cool down to room temperature using a thermocycler. 3. Dilute the annealed oligos 20 folds to the final concentration of 100 nM with ddH2O.

Fig. 2 (a) Sequence of the cloning sites of the pACRISPR plasmid. (b) Sequence of the primers designed for spacer insertion in the pACRISPR and pnCasPA-BEC plasmid

CRISPR/Cas9-based Genome Editing

7

4. Mix the following components in a sterile PCR tube for Golden Gate assembly: Component

Volume

10× T4 DNA ligase buffer (NEB)

1 μL

The pACRISPR plasmid (100 ng/μL)

1 μL

Annealed oligos (100 nM)

1 μL

T4 DNA ligase (NEB)

0.5 μL

BsaI-HF (NEB)

0.5 μL

ddH2O to 10 μL

5. Run the reaction in a thermocycler with the following parameters: Segment

Temperature

Time

Cycles

1

37 °C 16 °C

3 min 4 min

25

2

80 °C

15 min

1

3

10 °C

Forever

1

6. Transform the 10 μL Golden Gate assembly product into 100 μL E. coli DH5α competent cells. Plate the cells on the LB agar plate containing 50 μg/mL carbenicillin and incubate at 37 °C overnight. 7. Pick colonies from the plate and incubate in 5 mL LB medium containing 50 μg/mL carbenicillin at 37 °C overnight. 8. Extract the constructed pACRISPR-XX_spacer plasmid using plasmid mini-prep kit following the manufacturer’s instructions, and sequence the plasmid to confirm that the spacer has been successfully inserted. 3.1.2 Assembly of the Repair Template into the pACRISPR-XX_spacer Plasmid

1. Digest the pACRISPR-XX_spacer plasmid with XbaI and XhoI in a sterile PCR tube: Component

Volume

10× CutSmart buffer (NEB)

5 μL

2 μg pACRISPR-XX_spacer plasmid

xx μL

XbaI

2 μL

XhoI

2 μL

ddH2O to 50 μL

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Weizhong Chen and Quanjiang Ji

2. Incubate the reaction at 37 °C for 2–3 h. Purify the digested plasmid by using the PCR product purification kit for subsequent steps. 3. Select ~500 bp DNA sequences upstream and downstream of the target gene, respectively (see Note 2). The two DNA fragments were amplified using the genomic DNA of the P. aeruginosa strains as PCR template. The PCR products were purified by using the PCR product purification kit. 4. Mix the following components in a sterile PCR tube for Gibson assembly reaction (see Note 3): Component

Volume

NEBuilder HiFi DNA Assembly Master Mix (NEB)

5 μL

20 fmol XbaI/XhoI digested pACRISPR-XX_spacer plasmid xx μL 20 fmol DNA fragment upstream of the target gene

xx μL

20 fmol DNA fragment downstream of the target gene

xx μL

ddH2O to 10 μL

5. Incubate the reaction at 50 °C for 30 min. 6. Transform the 10 μL reaction solution into 100 μL E. coli DH5α competent cells. Plate the cells on the LB agar plate containing 50 μg/mL carbenicillin and incubate the plate at 37 °C overnight. 7. Pick colonies from the plate for plasmid extraction. Sequence the constructed plasmid pACRISPR-XX to confirm that the repair arms have been successfully assembled. 3.2 Genome Editing in P. aeruginosa Strains

1. Pick a single colony of P. aeruginosa strain and incubate in 100 mL LB medium at 37 °C overnight.

3.2.1 Preparation of P. aeruginosa Electrocompetent Cells

3. Wash the cell pellet twice with 40 mL of sterile ice-cold 10% v/v glycerol.

3.2.2 Prepare P. aeruginosa Electrocompetent Cells Containing the pCasPA Plasmid

2. Harvest the cells by centrifugation at 4 °C for 5 min at 6000g.

4. Resuspend the cells with 1 mL of 10% v/v glycerol. 50 μL aliquots of the cells were quenched in liquid nitrogen and stored in -80 °C for the subsequent experiments. 1. Take a tube of wild-type P. aeruginosa electrocompetent cells and thaw it on ice for 5 min. 2. Add ~500 ng pCasPA plasmid into the cells and mix well. Transfer the mixture into a 1 mm electroporation cuvette. 3. Pulse the cells at Gene Pulser Xcell™ Electroporation System (Bio-Rad) with the parameters of 2100 V, 200 Ω, 25 μF.

CRISPR/Cas9-based Genome Editing

9

4. Immediately add 1 mL of fresh LB medium into the cuvette after electroporation. Then transfer the cells into a 1.5 mL sterile Eppendorf tube. Shake at 37 °C for 1 h. 5. Plate the cells on the LB agar plate containing 100 μg/mL tetracycline. Incubate the plate at 37 °C overnight. 6. Pick a colony from the plate and incubate in 100 mL LB medium containing 100 μg/mL tetracycline at 37 °C overnight. 7. The next day, add 1 mL 20%w/v L-arabinose into the culture. Shake at 37 °C for 2–3 h. 8. Harvest the cells by centrifugation at 4 °C for 5 min at 6000g. Wash the cell pellet twice with 40 mL of sterile ice-cold 10% v/v glycerol. 9. Resuspend the cells with 1 mL of 10% v/v glycerol. Dispense the cells into 50 μL aliquots in 1.5 mL sterile Eppendorf tubes for the subsequent experiments (see Note 4). 3.2.3 pCasPA/pACRISPRmediated Genome Editing in P. aeruginosa

1. Add 1 μg constructed pACRISPR-XX plasmid into the electrocompetent cells of P. aeruginosa strain containing the pCasPA plasmid. Transfer the mixture into a 1 mm electroporation cuvette. 2. Pulse the cells at Gene Pulser Xcell™ Electroporation System (Bio-Rad) with the parameters of 2100 V, 200 Ω, 25 μF. 3. Immediately add 1 mL of fresh LB medium into the cuvette after electroporation. Then transfer the cells into a 1.5 mL sterile Eppendorf tube. Shake at 37 °C for 1–2 h. 4. Plate the cells on the LB agar plate containing 100 μg/mL tetracycline and 150 μg/mL carbenicillin. Incubate the plate at 37 °C overnight. 5. Screen the successfully edited colonies by PCR and sequencing (see Note 5).

3.2.4

Plasmid Curing

1. Pick a colony of P. aeruginosa containing the desired modification. Incubate it in fresh LB medium until the growth is evident. 2. Diluted the culture for 104 folds with fresh LB medium. Plate 100 μL diluted culture onto the LB agar plate supplemented with 5% w/v sucrose (see Note 6). Incubate the plate at 37 °C overnight. 3. Pick 4–6 individual colonies and streak them onto three different LB agar plates (no antibiotics, 100 μg/mL tetracycline, and 150 μg/mL carbenicillin). If the plasmid has been successfully cured, the colonies will grow normally on the plate without antibiotics, but could not grow on the plate in the presence of tetracycline or carbenicillin.

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Weizhong Chen and Quanjiang Ji

3.3 pnCasPA-BECmediated Cytidine Base Editing in P. aeruginosa

1. Select a suitable 20-nt spacer sequence immediately upstream of the 5′-NGG-3′ PAM in the target site (see Note 7). Synthesize the two oligos as shown in Fig. 2b. 2. Insert the spacer into the pnCasPA-BEC plasmid via Golden Gate assembly (see Note 8). The constructed pnCasPA-BECXX plasmid was screened on an LB agar plate containing 30 μg/mL gentamycin and verified by sequencing. 3. Electroporate the editing plasmid pnCasPA-BEC-XX into wild-type P. aeruginosa electrocomplement cells with the parameters of 2100 V, 200 Ω, 25 μF, 1 mm cuvette. 4. Immediately add 1 mL of fresh LB medium into the cuvette after electroporation. Then transfer the cells into a 1.5 mL sterile Eppendorf tube and shake at 37 °C for 1 h. 5. The cells were plated onto the LB agar plate containing 30 μg/ mL gentamycin. Incubate the plate at 37 °C overnight. 6. Pick 2–4 individual colonies. Amplify the DNA regions which cover the editable sites using the genomic DNA of colonies as the PCR template. Sequence the PCR products to verify whether the colonies were successfully edited (see Note 9). 7. The pnCasPA-BEC plasmid can be rapidly cured by plating the cells on an LB agar plate supplemented with 5% w/v sucrose. After plasmid curing, pick 4–6 individual colonies and streak them onto LB agar plates in the absence or presence of 30 μg/ mL gentamycin. If the plasmid has been cured, the colonies will grow normally on the plate without gentamycin, but could not grow on the plate containing 30 μg/mL gentamycin.

4

Notes 1. The GC ratio of the spacer sequence usually is 40–60%. Please note that the NGG PAM is not included in the spacer. 2. The repair template should contain two repair arms that are homologous to the upstream and downstream of the target locus, respectively. For Gibson assembly, the repair template should have a 20–40 bp overlap with the pACRISPRXX_spacer plasmid. To guarantee the efficiency of homologous recombination, the length of repair arms should be 500 bp or longer. Short repair arms ( AC or CC and avoiding using GC. 8. The cloning sites of the pnCasPA-BEC plasmid are the same as that of the pACRISPR plasmid (Fig. 2a). Therefore, insertion of spacers into pnCasPA-BEC follows the same procedure as that of pACRISPR. The pnCasPA-BEC plasmid can execute the editing after the insertion of a spacer, without using a repair template. 9. If the Sanger sequencing result presents a nested peak at the editing site, indicating that only a portion of cells was successfully edited, please streak the cells on the LB agar plate and pick single colonies again. Then the pure colony with the desired modification would be obtained.

Acknowledgments This work was financially supported by the National Natural Science Foundation of China (grant nos. 91753127, 21922705, 22077083 to Q. J, 21907066 to W. C), National Key R&D Program of China Grant (no. 2017YFA0506800 to Q. J), and the Shanghai Science and Technology Committee Rising-Star Program (grant no. 19QA1406000 to Q. J). References 1. Horvath P, Barrangou R (2010) CRISPR/Cas, the immune system of bacteria and archaea. Science 327(5962):167–170 2. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al (2013) Multiplex genome

engineering using CRISPR/Cas systems. Science 339(6121):819–823 3. Bortesi L, Fischer R (2015) The CRISPR/ Cas9 system for plant genome editing and beyond. Biotechnol Adv 33(1):41–52

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4. DiCarlo JE, Norville JE, Mali P, Rios X, Aach J, Church GM (2013) Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res 41(7):4336–4343 5. Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA (2013) RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 31(3):233–239 6. Chen W, Zhang Y, Yeo WS, Bae T, Ji Q (2017) Rapid and efficient genome editing in Staphylococcus aureus by using an engineered CRISPR/ Cas9 system. J Am Chem Soc 139(10): 3790–3795 7. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E (2012) A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816–821 8. Wiedenheft B, Sternberg SH, Doudna JA (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature 482(7385): 331–338 9. Peters JM, Silvis MR, Zhao D, Hawkins JS, Gross CA, Qi LS (2015) Bacterial CRISPR: accomplishments and prospects. Curr Opin Microbiol 27:121–126

10. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR (2016) Programmable editing of a target base in genomic DNA without doublestranded DNA cleavage. Nature 533(7603): 420–424 11. Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI et al (2017) Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551(7681):464–471 12. Zhao D, Li J, Li S, Xin X, Hu M, Price MA et al (2021) Glycosylase base editors enable C-to-A and C-to-G base changes. Nat Biotechnol 39(1):35–40 13. Kurt IC, Zhou R, Iyer S, Garcia SP, Miller BR, Langner LM et al (2021) CRISPR C-to-G base editors for inducing targeted DNA transversions in human cells. Nat Biotechnol 39(1): 41–46 14. Chen W, Zhang Y, Zhang Y, Pi Y, Gu T, Song L et al (2018) CRISPR/Cas9-based genome editing in Pseudomonas aeruginosa and cytidine deaminase-mediated base editing in Pseudomonas species. iScience 6:222–231

Chapter 2 Investigating Pseudomonas aeruginosa Gene Function During Pathogenesis Using Mobile-CRISPRi Michelle A. Yu, Amy B. Banta, Ryan D. Ward, Neha K. Prasad, Michael S. Kwon, Oren S. Rosenberg, and Jason M. Peters Abstract CRISPR interference (CRISPRi) is a robust gene silencing technique that is ideal for targeting essential and conditionally essential (CE) genes. CRISPRi is especially valuable for investigating gene function in pathogens such as P. aeruginosa where essential and CE genes underlie clinically important phenotypes such as antibiotic susceptibility and virulence. To facilitate the use of CRISPRi in diverse bacteria— including P. aeruginosa—we developed a suite of modular, mobilizable, and integrating vectors we call, “Mobile-CRISPRi.” We further optimized Mobile-CRISPRi for use in P. aeruginosa mouse models of acute lung infection by expressing the CRISPRi machinery at low levels constitutively, enabling partial knockdown of essential and CE genes without the need for an exogenous inducer. Here, we describe protocols for creating Mobile-CRISPRi knockdown strains and testing their phenotypes in a mouse pneumonia model of P. aeruginosa infection. In addition, we provide comprehensive guide RNA designs to target genes in common laboratory strains of P. aeruginosa and other Pseudomonas species. Key words CRISPR-Cas9, CRISPRi, Essential genes, Murine pneumonia model, Systems biology, Pseudomonas putida, Pseudomonas syringae

1

Introduction CRISPRi—the use of a catalytically inactive variant of the Cas9 nuclease (dCas9) and single guide RNAs (sgRNAs) to repress gene expression ([1, 2]; Fig. 1a)—has advantages over classical genetic approaches such as gene deletion or transposon mutagenesis for characterizing essential and CE gene phenotypes. Essential genes cannot be deleted by definition [3, 4] and only tolerate transposon insertions in non-essential domains [5–7] while CE genes can be disrupted only under conditions in which they are not essential. In contrast, CRISPRi can target both essential and CE genes via partial knockdown using the following (non-mutually exclusive) approaches: (1) expressing CRISPRi components under

Giovanni Bertoni and Silvia Ferrara (eds.), Pseudomonas aeruginosa: Methods and Protocols, Methods in Molecular Biology, vol. 2721, https://doi.org/10.1007/978-1-0716-3473-8_2, © The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature 2024

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Michelle A. Yu et al. A

CRISPRi

B sgRNA

dCas9

Mobile-CRISPRi construct dCas9 promoter

RNAP

P1

Tn7 R

gmR

sgRNA

P2

P3

dcas9

Tn7 L

spacer

nascent RNA

C

Triparental mating

glmS

E. coli CRISPRi transposon donor

CRISPRi

Pseudomonas recipient E. coli Tn7 transposase donor

Pseudomonas CRISPRi chromosomal insertion

D

Lung homogenization Infection progression

Intratracheal instillation of Pseudomonas

Pseudomonas enumeration

Fig. 1 Pseudomonas CRISPRi experimental overview. (a) Mechanism of CRISPRi repression. A dCas9-sgRNA complex binds to a specific DNA sequence and sterically blocks transcription elongation by RNA polymerase (RNAP), thereby reducing gene expression. (b) Modular Mobile-CRISPRi system. The Mobile-CRISPRi construct encodes antibiotic resistance, sgRNA, and dCas9 expression cassettes on a Tn7 transposon. The modular design enables these components and their promoters to be exchanged as necessary by restriction enzyme digest followed by Gibson assembly to optimize Mobile-CRISPRi for a particular organism or experiment. The version detailed in this protocol for constitutive expression in Pseudomonas aeruginosa encodes gentamicin resistance (gmR) and one of three constitutive dCas9 promoters of varying strength (P1, P2, P3). DNA encoding the 20-nt spacer region of the sgRNA can be cloned in between BsaI sites. (c) Mobile-CRISPRi strain construction. CRISPRi-expressing strains are constructed by triparental mating of E. coli donor strains (one harboring a Mobile-CRISPRi plasmid and another harboring a plasmid expressing the Tn7 transposase) with a Pseudomonas recipient. The CRISPRi expression cassette will be stably incorporated onto the Pseudomonas chromosome at the Tn7 att site located downstream from glmS. (d) P. aeruginosa CRISPRi murine infection experiment. Diluted P. aeruginosa culture is used to inoculate mouse lungs via intratracheal instillation. P. aeruginosa present in infected lungs are enumerated by homogenization followed by dilution and counting of colonies grown on agar plates. (Panel D was partially created with BioRender.com)

inducible promoters and varying inducer concentration (e.g., xylose [8], IPTG [9], aTc [10]), (2) deliberately inserting mismatches between sgRNA spacer sequences and their target genes (i.e., Mismatch-CRISPRi [11–13]), and (3) expressing CRISPRi components using a series of weak, constitutive promoters ([14]; Fig. 1b). The 20 nucleotide sgRNA spacer not only determines the

Mobile-CRISPRi in P. aeruginosa

15

gene targeted by CRISPRi, but also serves as a convenient DNA barcode for monitoring the abundance of individual knockdown strains in a pooled context [15]. Due to its advantages, CRISPRi is quickly becoming the preeminent tool for bacterial gene function discovery in many contexts, including pathogenesis. CRISPRi libraries have been used at the genome scale to study essential gene interactions with antibiotics [8], identify host factors involved in phage predation [16, 17], explore cross-strain differences in essentiality [18], and characterize morphological defects associated with essential gene depletion [8, 19], among other uses (reviewed in [20]). Recent work has highlighted the value of CRISPRi in characterizing and identifying essential and CE genes during pathogenesis; these studies range from simple proof-of-principle experiments [14] to genome-scale analysis of virulence-associated genes [21], with some even exploring the effects of multiplexed knockdowns [22]. Few studies have utilized CRISPR platforms to screen for pathogen virulence factors in the context of mammalian hosts [21, 23]. Yet, studies that combine host-pathogen responses with functional gene analyses are paramount to understanding how bacteria adapt to host environments and developing novel antimicrobial targets. To deliver CRISPRi to diverse bacteria and to enable our own studies of pathogenesis in P. aeruginosa, we developed Mobile-CRISPRi ([14, 24]; Fig. 1b, c). Mobile-CRISPRi has several advantages over other CRISPRi delivery and expression systems including: (1) modularity; individual components such as sgRNA spacers or the dcas9 promoter can be swapped cleaving the vectors at flanking restriction recognition sites followed by ligation or Gibson assembly [25] of new components (Fig. 1b), (2) transfer by mating (i.e., conjugation), and (3) stable integration into the chromosome of the recipient bacterium (Fig. 1c). For the Tn7-based version of Mobile-CRISPRi used in P. aeruginosa, CRISPRi components integrate site-specifically into the genome downstream of the essential gene, glmS, but do not disrupt glmS function [26]. Integration of CRISPRi is key for pathogenesis experiments—Mobile-CRISPRi is inherited stably without selection and is not subject to copy number variation that would otherwise generate experimental noise. Our P. aeruginosa-optimized Mobile-CRISPRi systems achieve partial knockdown of gene expression by using weak, constitutive promoters to drive dcas9 expression ([14]; Fig. 1b). Because these promoters are of varying strength (P1 is weakest and P3 is strongest), one can produce a knockdown gradient by using the complete series of vectors in CRISPRi; this enables studies of the relationship between knockdown and fitness in the context of pathogenesis without the need for exogenous inducer that is difficult to deliver in controlled amounts. Finally, our Mobile-CRISPRi systems are non-toxic to P. aeruginosa when not targeting essential and CE genes and show

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no growth defects in mouse infection experiments (i.e., CFU recovery from lungs; Fig. 1d). Here we present protocols for defining new gene knockdown targets using Mobile-CRISPRi and characterizing knockdown strain phenotypes in a mouse pneumonia model. We provide comprehensive sgRNA spacer designs for the most widely studied Pseudomonas genomes, including other Pseudomonas species such as P. putida and P. syringae (all “popular” genomes from the pseudomonas.com database) as interactive spreadsheets (available at https://github.com/ryandward/Pseudomonas_sgRNA); these spreadsheets contain oligo sequences for cloning sgRNA spacers by annealing as well as links to NCBI tracks that illustrate the location of sgRNA targets along genes. We describe the process of generating Mobile-CRISPRi vectors to specify gene targets, starting from cloning annealed oligos into Mobile-CRISPRi to define new sgRNA spacers and then conjugating Mobile-CRISPRi vectors into P. aeruginosa recipient strains. Next, we provide a protocol for our P. aeruginosa acute lung infection model that utilizes intratracheal installation of a defined inoculum of CFU into wild-type mice, followed by lung collection, homogenization, and plating at 24 h to quantify CFUs as a measure of virulence. Strains containing CRISPRi knockdown of an essential or CE gene will exhibit reduced CFU recovery. Furthermore, this infection protocol translates easily to study pathogenesis in other murine models of disease such as immunocompromised mice.

2

Materials All reagents, media, and solutions should be prepared with purified deionized water (dH2O) (e.g., Milli-Q). Solutions should be sterilized, as indicated below, either by autoclaving 20–50 min, liquid cycle or by filtration through a 0.2 μm filter (syringe filter, steriflip, or bottle top filter). Disposable supplies such as pipet tips, microfuge tubes, petri dishes, etc. should be sterile.

2.1

Bacterial Strains

1. Bacterial strains used in this protocol can be found in Table 1. 2. Escherichia coli electrocompetent cells may be prepared according to the protocol of your choice or using a protocol such as the one in [27]. The strains used in this study are not currently commercially available as competent cells.

2.2

Plasmids

1. Plasmids used in this protocol can be found in Table 2. 2. Plasmids can be ordered from Addgene (https://www. addgene.org/), Watertown, MA. 3. Plasmid maps and full sequences can be downloaded from Addgene using the accession numbers in the table.

Mobile-CRISPRi in P. aeruginosa

17

Table 1 Strains Strain

JMP #

Genotype

Media

Comment

Ref.

Pseudomonas aeruginosa UCBPPPA14

sJMP012 Wild type

LB

P. aeruginosa PA14 recipient strain

[30]

E. coli pir+ ‘cloning strain’

[31]

Escherichia coli sJMP424 lacIq, rrnB3, DElacZ4787, hsdR514, DE LB + DAP E. coli pir+ WM6026 (araBAD)567, DE(rhaBAD)568, rph‘mating 1 att-lambda::pAE12-del (oriR6K/cat:: strain’ frt5), D4229(dapA)::frt(DAP-), D (endA)::frt, uidA(DMluI)::pir(wt), attHK::pJK1006::D1/2(DoriR6Kcat::frt5, DtrfA::frt)

[32]

Escherichia coli sJMP146 Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), LB BW25141 Δ(phoB-phoR)580, _-, galU95, ΔuidA3::pir+, recA1, endA9(Dins):: FRT, rph-1, Δ(rhaD-rhaB)568, hsdR514

2.3 sgRNA Spacer Sequences

1. sgRNA spacers against all target genes in multiple Pseudomonas genomes have been designed and are available for download at: https://github.com/ryandward/Pseudomonas_sgRNA. To design sgRNA spacers for additional genomes not included in this set, please see detailed information in [27] (see Note 1). 2. New sgRNA spacers are encoded in the Mobile-CRISPRi system by annealing two oligonucleotides such that they encode the spacer and have 4 nt overhangs for cloning into the BsaI site of the Mobile-CRISPRi vector. See Fig. 2 for an illustration of this technique and for an example of the oligonucleotides needed to encode a spacer for your target gene. 3. Oligonucleotides are standard purity 24-mers from Integrated DNA Technology, Coralville IA.

2.4 Bacterial Media and Additives

1. Lysogeny broth (LB): Dissolve 10 g tryptone, 5 g yeast extract, and 5 g NaCl in dH2O. Sterilize by autoclaving. 2. For plates, add agar to 1.5% prior to autoclaving. Cool to 50 °C before mixing in additives and pouring plates. 3. SOC medium: Dissolve 4 g tryptone, 1 g yeast extract, 0.1 g NaCl, and 500 μL 1 M KCl in 200 mL H2O in a bottle with a stir bar and sterilize by autoclaving. After cooling, mix in 1 mL sterile 1 M MgCL2 and 3.6 mL sterile 20% w/v glucose. Aliquot 10 mL/tube and store between -20 and 25 °C.

JMP#

pJMP2633 134647

pJMP2635 134657

pJQ48

pJQ49

Trc (no operator) Trc (no operator)

ampR, gmR

Trc (no operator)

N/A

ampR, gmR

amp , gm

R

sgRNA Promoter

P3 (Anderson BBa_J23115)

P2 (Anderson BBa_J23114)

P1 (Anderson BBa_J23117)

N/A

dCas9 Promoter

Hsa Spy dcas9::3Xmyc

Hsa Spy dcas9::3Xmyc

Hsa Spy dcas9::3Xmyc

N/A

dCas9

BsaI site for cloning new sgRNAs

BsaI site for cloning new sgRNAs

BsaI site for cloning new sgRNAs

Expresses Tn7 transposase

Comment

[14]

[14]

[14]

[24]

Ref.

All plasmids have R6K ori (replicate only in pir+ strain), Hsa Spy::3Xmyc dCas9 is human codon-optimized, ampR (100 μg/mL ampicillin or carbenicillin), gmR (gentamicin, 15 μg/mL (E. coli replicating plasmid), 30 μg/mL (integrated onto Pseudomonas chromosome))

pJMP2631 134646

pJQ47

R

ampR, N/A

Addgene Antibiotic # resistance

pTn7C1 pJMP1039 119239

Name

Table 2 Plasmids

18 Michelle A. Yu et al.

Mobile-CRISPRi in P. aeruginosa BsaI

BsaI

19

BsaI

A. Digest plasmid promoter

B. Anneal oligos

cloning site

dCas9 handle (constant)

5’-TAGTAACTTTCAGTTTAGCGGTCT-3’ |||||||||||||||||||| 3’-TTGAAAGTCAAATCGCCAGACAAA-5’

C. Ligate promoter

spacer

dCas9 handle (constant)

sgRNA

Fig. 2 sgRNA spacer cloning. Shown here is the sgRNA module from Mobile-CRISPRi. (a) The Mobile-CRISPRi vector is digested with BsaI. (b) Oligonucleotides are annealed so as to form overhangs compatible with the BsaI-digested vector. (c) Annealed oligonucleotides and BsaI-digested vector are ligated to encode the complete sgRNA consisting of the new (variable) spacer sequence and the (constant) dCas9 handle. This figure depicts an mRFP targeting spacer, but 20 nt spacer sequences targeting any gene of interest can be cloned using this protocol. All BsaI recognition sites are lost in the cloning procedure. The middle BsaI site will further digest the unneeded portion of the vector to reduce the cloning background

4. Pseudomonas Isolation Agar (PIA, Sigma-Aldrich 17208). 5. Ampicillin (1000× stock): 100 mg/mL in dH2O. Filter, sterilize, and store aliquots at -20 °C. 6. Gentamicin (500× stock): 15 mg/mL in dH2O. Filter, sterilize, and store aliquots at -20 °C. 7. 30 mM Diaminopimelic acid (DAP) (100× stock): 0.285 g/ 50 mL dH2O. Filter, sterilize, and store at 4 °C. 8. 50% glycerol: Mix 50 mL glycerol with 50 mL dH2O and sterilize by autoclave. Add to cultures at a final concentration of 15% to make stocks that can be stored at -80 °C. 2.5 Equipment and Supplies for Microbiology and Molecular Biology

1. General laboratory disposables: pipet tips, microfuge tubes, pipettes, culture tubes, petri dishes, electroporation cuvettes (1 mm gap), sterile inoculation loops, etc. 2. General laboratory equipment: Centrifuge, microcentrifuge, pipettors, filter forceps, electroporator, incubator and incubator-shaker (or roller drum) at 37 °C, heated dry block (or thermal cycler) capable of incubating at 37 °C, 65 °C, 80 ° C, and 95 °C, etc. 3. Cellulose filters (e.g., MF-Millipore HAWG01300) for conjugation.

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2.6 Nucleic Acid Purification Kits

1. DNA spin purification kit (e.g., Zymo Research D4003).

2.7 Enzymatic Reactions

1. BsaI-HF-v2 restriction enzyme and 10× Cutsmart buffer (New England Biolabs R3733).

2. Plasmid DNA miniprep kit (e.g., Thermo GeneJet kit K0502).

2. T4 DNA ligase (e.g., New England Biolabs M0202). 3. 1 mM ATP (e.g., Thermo R0441, diluted 100×). 4. 100 mM DL-dithiothreitol (DTT, Sigma 9779): Dissolve 0.154 g DTT (MW 154.3) in a final volume of 10 mL dH2O, sterilize by filtration, and store 1 mL aliquots at -20 °C. 2.8 Equipment and Supplies for Mouse Infection and Analysis

1. Pipet tips.

2.8.1 General Laboratory Disposables

4. Sterile test tubes for lung collection (Fisher Scientific 149595).

2. Microfuge tubes. 3. Pipets. 5. Petri dishes. 6. Sterile L-spreaders.

2.8.2 General Laboratory Equipment

1. Centrifuge. 2. Microcentrifuge. 3. Pipettors. 4. Incubator and incubator-shaker (or roller drum) at 37 °C, heated dry block. 5. PCR thermocycler. 6. Tissue homogenizer (Kinematica AG Polytron PT 1200 E, Kinematica 11010025). 7. BSL2 hood. 8. Rodent weighing scale.

2.8.3 General Mouse Facility Equipment

1. Rodent barrier facility. 2. Cages. 3. Food. 4. Water. 5. Designated BSL2 animal procedure space.

2.8.4 Surgical and Anesthesia Equipment

1. Stevens tenotomy curved scissors. 2. Metzenbaum curved scissors. 3. Dumont tweezers #5. 4. Offset flat-tip forceps. 5. Iris curved forceps. 6. Rochester-Carmalt hemostatic forceps.

Mobile-CRISPRi in P. aeruginosa

21

7. Polyethylene PE-10 tubing (Intramedic 427401). 8. 0.5 mL U-100 BD insulin syringe (28G, 1/2″, Fisher Scientific 1482679). 9. Mouse intubation platform [28]. 10. Isoflurane rodent anesthesia chamber. 11. Single gooseneck fiber-optic light (Cole Parmer Fiber-Lite, model 974500). 12. OptiVisor 2.5× (working distance 8″; alternative 3.5× with working distance 4″ (VWR 100499-546). 13. Rodent thermometer (Fisherbrand 150783A). 14. Masking tape. 2.9 Reagents for Mouse Infection and Analysis

1. Sterile PBS. 2. Evans blue (MP Biomedicals 314136).

2.9.1 Reagents for Mouse Infection 2.9.2 Reagents for Mouse Anesthesia

1. Isoflurane (Piramal Critical Care 66794-017-25). 2. Ketamine (Henry Schein 010177). 3. Xylazine (Henry Schein 033198).

2.10

Mice

1. 10–20 C57BL/6 WT mice, age 8–12 weeks (Jackson Labs 000664) per experiment (see Note 2). 2. Mice to practice direct visual intratracheal instillation technique (see Note 3 and Fig. 3). 3. Mouse models relevant to the disease of interest.

3

Methods

3.1 Cloning sgRNA Spacers into MobileCRISPRi Vectors

Mobile-CRISPRi is a modular system encoding the components needed for CRISPR-mediated gene repression [24]. A version of this system has been specifically developed for use in Pseudomonas infection studies [14]. The Mobile-CRISPRi vector encodes dCas9, an sgRNA, and an antibiotic resistance marker between Tn7 transposon ends (see Fig. 1b). This protocol describes how to construct a Mobile-CRISPRi vector encoding an sgRNA spacer targeting your gene of interest. Briefly, two oligonucleotides are designed so that when annealed they encode the spacer as well as overhangs enabling cloning into the Mobile-CRISPRi vector (see Fig. 2). This plasmid then acts as a donor for transfer by Tn7 transposition of the Mobile-CRISPRi system onto the Pseudomonas chromosome at the attTn7 site just downstream of glmS (see Fig. 1c).

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Fig. 3 Intratracheal instillation of mice with 50 μL methylene blue dye. (a) Surgical tools from left to right: Stevens tenotomy curved scissors, Metzenbaum curved scissors, Dumont tweezers #5, offset flat tip forceps, Iris curved forceps, Rochester-Carmalt hemostatic forceps, 0.5 mL U-100 BD insulin syringe (28G, 1/2″). (b) Syringe with PE-10 tubing with marked tip to optimize visualization. (c) Intubation platform for intratracheal instillation. (d) Fiber-optic lamp positioned to illuminate the trachea. (e) Tongue retraction using the offset flat tip forceps to visualize the trachea with minimal soft tissue injury to the tongue. (f) Insertion of the syringe into the trachea. (g) Thoracotomy exposing heart and lung visualized under a dissecting microscope. (h) Three sets of dissected lungs, showing bilateral (black arrows) or unilateral (white arrow) distribution of dye. Unilateral distribution can occur if the syringe is advanced too distally into the right or left mainstem bronchus and is suboptimal. (i) Distribution of dye into the stomach indicates that the syringe entered the esophagus and not the trachea 3.1.1 Preparation of BsaI-digested MobileCRISPRi Plasmid

1. Digest 1 μg Mobile-CRISPRi plasmid DNA with BsaI: 5 μL 10× cutsmart buffer (NEB), 10 μL plasmid DNA (100 μg/ mL), 1 μL BsaI-HF-v2 (NEB), 34 μL dH2O (adjust the volumes of dH2O and DNA as necessary based on the concentration of the DNA for a total reaction volume of 50 μL) (see Note 4).

Mobile-CRISPRi in P. aeruginosa

23

2. Incubate at 37 °C, 2–8 h. 3. Heat to inactivate the enzyme by incubating at 80 °C for 20 min. 4. Purify the digested DNA with a DNA spin purification kit according to the manufacturer’s protocol, eluting in ~10 μL for a final concentration of ~25–50 ng/μL (see Note 5). 3.1.2 Annealing of sgRNA Spacer-encoding Oligonucleotides

1. Mix 5 μL 10× Cutsmart Buffer (NEB), 1 μL each top and bottom oligonucleotides (100 μM), and 43 μL dH2O (total reaction volume 50 μL). 2. Incubate at 95 °C for 5 min to denature DNA. 3. Cool at room oligonucleotides.

temperature

for

15

min

to

anneal

4. Dilute 1:40 in dH2O (see Note 6). 3.1.3 Ligation of Annealed Oligonucleotides and BsaI-digested Vector

Set up one reaction for each set of oligonucleotides plus a vectoronly negative control. 1. For each ligation, mix: 1 μL T4 DNA ligase buffer (NEB), 1 μL 100 mM DTT, 1 μL 1 mM ATP, 0.5 μL T4 DNA ligase (NEB), 2 μL 1:40 diluted oligonucleotides, 2 μL BsaI-digested purified Mobile-CRISPRi plasmid (~25–50 ng/μL), and 2.5 μL dH2O (total reaction volume 10 μL) (see Note 7). 2. Incubate at room temperature for 1–2 h to ligate. 3. Incubate at 65 °C for 15 min to inactivate the enzyme.

3.1.4

Transformation

1. Thaw electrocompetent E. coli BW25141 cells on ice for ~5 min. 2. Mix 50 μL cells and 1 μL ligation in a sterile 1.5 mL microfuge tube on ice. 3. Transfer the mixture to a 0.1 cm gap electroporation cuvette on ice (see Note 8). 4. Wipe moisture from outside of cuvette and pulse in an electroporator set to exponential decay pulse, 25 μF, 200 ohm, 1.8 kV (i.e., “Bacterial 1” preset on BioRad Gene Pulser Xcell or “EC1” on BioRad Micropulser). 5. Immediately remove the cuvette from the electroporator, add 800 μL SOC medium, mix with cells by pipetting, and transfer to a culture tube (see Note 9). 6. Incubate at 37 °C, 1 h, shaking at 250 rpm or rotating on a roller drum. 7. Plate various amounts of transformation on 2–3 prewarmed appropriate selective plates (e.g., LB agar + ampicillin for E. coli BW25141 and the Mobile-CRISPRi plasmid) and incubate at 37 °C, ~16 h.

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8. If colonies are not sufficiently isolated, restreak for isolation on appropriate selective plates and incubate at 37 °C, ~16 h. 3.1.5 Confirmation of Mobile-CRISPRi Plasmid Clones

1. Inoculate 5 mL LB + ampicillin medium in a culture tube with a single colony and incubate at 37 °C, ~16 h, shaking at 250 rpm or rotating on a roller drum. 2. Extract plasmid DNA from the entire 5 mL culture using a kit following the manufacturer’s instructions (see Note 10). 3. Sequence the region of the plasmid encoding the new sgRNA spacer. 4. Store plasmid DNA at -20 °C. Stock strains containing plasmids for later use in LB + ampicillin + 15% glycerol and store at -80 °C.

3.2 Transferring Mobile-CRISPRi to Pseudomonas by Tn7 Transposition

After constructing Mobile-CRISPRi plasmid(s) encoding sgRNA spacers targeting your gene(s) of interest, they will need to be transferred to an E. coli WM6026 donor strain before conjugal transfer to a recipient Pseudomonas strain (see Note 11). Tri-parental mating of a Mobile-CRISPRi donor strain, a Tn7 transposase donor strain, and the recipient Pseudomonas will enable Tn7 transposition of the Mobile-CRISPRi system onto the Pseudomonas chromosome at the attTn7 site just downstream of glmS (see Fig. 1c). Final selection on plates lacking DAP will prevent the donor E. coli strains from growing and the plasmids will be lost because they cannot replicate in a host lacking the pir gene. Selection for the antibiotic resistance marker on the transposon (gentamicin resistance) will result in the growth only of the Pseudomonas recipients with an integrated Mobile-CRISPRi system.

3.2.1 Construction of Donor Strains for Tn7 Transposition

You will need to create at least three strains: one with your newly cloned Mobile-CRISPRi plasmid (encoding spacer targeting your gene of interest), one with the parent Mobile-CRISPRi plasmid (no spacer control; pJQ47-49), and one with a plasmid encoding the Tn7 transposase (pTn7C1). 1. Follow the transformation procedure in Subheading 3.1.4 except use E. coli WM6026 and 1 μL of plasmid DNA and select on LB + ampicillin (100 μg/mL) + DAP (300 μM) agar plates (see Note 12). Be sure to include DAP in the transformation recovery medium (i.e., SOC + DAP). 2. Stock strains for later use in LB + ampicillin + DAP +15% glycerol and store at -80 °C.

3.2.2 Tn7 Transposition of Mobile CRISPRi to the Pseudomonas aeruginosa Chromosome

1. Streak strains onto agar plates from -80 °C stocks and incubate ~14–18 h at 37 °C to obtain isolated colonies. For E. coli donor strains, use LB + ampicillin (100 μg/mL) + DAP (300 μM) agar plates and for the P. aeruginosa recipient strain, use LB (see Note 12).

Mobile-CRISPRi in P. aeruginosa

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2. Inoculate 5 mL medium in a culture tube with a single colony and grow to saturation by incubating ~14–18 h at 37 °C with aeration (shaking at 250 rpm or on drum roller). For E. coli transposon and transposase donor strains, use LB + ampicillin (100 μg/mL) + DAP (300 μM) and for P. aeruginosa recipient strain, use LB. 3. Add 700 μL LB to a sterile microfuge tube. Add 100 μL each of the 3 cultures for a total volume of 1 mL. Also set up three negative controls by leaving out either recipient, transposon donor, or transposase donor (see Note 13). 4. Centrifuge at 7000 × g for 2 min. 5. Remove media by pipetting and resuspend the pellet in 1 mL of LB. Centrifuge again at 7000 × g for 2 min. 6. Repeat the above wash step once. 7. Remove the supernatant and resuspend the pellet in 30 μL of LB. Pipet the cells onto a cellulose filter placed on a pre-warmed LB + 300 μM DAP plate. Incubate for ~5–24 h at 37 °C (see Notes 14 and 15). 8. Using ethanol and flame-sterilized tweezers, transfer the filter to a sterile microcentrifuge tube containing 200 μL of LB. Vortex ~15 s to dislodge the cells from the filter and resuspend in media. 9. Dilute suspension microcentrifuge tube.

1:100

in

LB

in

a

sterile

10. Plate 100 μL undiluted and 1:100 diluted cell suspension on LB + 30 μg/mL gentamicin agar medium that will select for the Mobile-CRISPRi transposon-encoded antibiotic resistance gene and recipient but will not support the growth of the donor E. coli strains due to lack of DAP. Adjust the amount plated or restreak to obtain isolated colonies (see Note 16). 11. Stock strains for later use in LB + 15% glycerol and store at 80 °C. 3.3 Testing Gene Essentiality in a Murine Lung Infection Model

Gene essentiality in vivo can be assessed based on the recovery rate of the Mobile-CRISPRi Pseudomonas strains relative to wild-type Pseudomonas and Mobile-CRISPRi control (non-targeting sgRNA) Pseudomonas strain following a murine lung infection (see Note 17). Pilot experiments must be conducted to establish the requisite bacterial burden for a productive infection and a suitable endpoint based on the progression of infection (see Notes 18 and 19). Before beginning an experiment, we suggest using Evans blue dye to test for proper instillation technique (see Note 3 and Fig. 3). Optimal technique will result in the distribution of blue dye into bilateral lungs without deposition into the stomach. Pitfalls to

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avoid include: (1) distribution of dye into a unilateral lung, which suggests that the syringe and PE-10 catheter have been introduced too distally into the right or left mainstem bronchus, (2) esophageal instillation which results in blue dye deposition in the stomach, and (3) oropharyngeal without intratracheal instillation, which suggests the syringe and catheter are too proximal and have not passed the larynx. 1. Streak Pseudomonas strains (wild-type, gene-targeting, and non-targeting controls) (see Note 17) onto LB agar plates from -80 °C stocks and incubate for ~14–18 h at 37 °C to obtain isolated colonies. 2. Inoculate 5 mL LB medium in a culture tube with a single colony and grow to saturation by incubating ~16 h at 37 °C with aeration (shaking at 250 rpm or on drum roller). 3. Dilute the cultures 1:100 in fresh LB media and grow to the mid-log phase (see Note 18). 4. Prepare the bacterial inocula for the mouse infection in microcentrifuge tubes by diluting the subcultures to the appropriate CFU/mL in 50 μL sterile PBS (see Note 19). 5. Record the weight and rectal temperature of mice then label subjects numerically using a permanent marker on the tail. 6. Place 10 animals at a time in the rodent isoflurane chamber. 7. Prepare an inoculum delivery syringe by attaching a 2 cm PE-10 tubing to the insulin syringe and needle. Pre-load this syringe with 50 μL of air to allow the entire inoculum to be delivered into the lungs. 8. When respiratory rate has slowed to