使用CRISPR/Cas9在大肠埃希杆菌中进行逐步多基因敲除

Enrico König; Francesca Zerbini; Ilaria Zanella; Davide Fraccascia; Guido Grandi

doi:10.21769/BioProtoc.2688

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Peer-reviewed

Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli

使用CRISPR/Cas9在大肠埃希杆菌中进行逐步多基因敲除

EK Enrico König ^*

FZ Francesca Zerbini ^*

IZ Ilaria Zanella ^*

DF Davide Fraccascia

GG Guido Grandi email

(*contributed equally to this work) 发布: 2018年01月20日第8卷第2期 DOI: 10.21769/BioProtoc.2688 浏览次数: 27511

评审: David CisnerosAlba BlesaTomas Aparicio

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Abstract

With the recent implementation of the CRISPR/Cas9 technology as a standard tool for genome editing, laboratories all over the world are undergoing one of the biggest advancements in molecular biology since PCR. The key advantage of this method is its simplicity and universal applicability for species of any phylum. Of particular interest is the extensively studied Gram-negative bacterium Escherichia coli, as it is considered as the workhorse for both research and industrial purposes. Here, we present a simple, robust and effective protocol using the CRISPR/Cas9 system in combination with the λ Red machinery for gene knockout in E. coli. Crucial in our procedure is the use of a double-stranded donor DNA and a curing strategy for removal of the guide RNA encoding plasmid that allows starting a new mutation after only two working days. Our protocol allows multiple, stepwise gene knockout strains with high mutagenesis efficiencies applicable for high-throughput approaches.

Keywords: Biotechnology (生物技术)

Synthetic biology (合成生物学)

High-throughput (高通量)

Genome editing (基因组编辑)

Double-stranded donor DNA (双链供体DNA)

Background

The Gram-negative bacterium Escherichia coli is one of the most important organisms for biotechnological engineering. It has been successfully implemented in a wide range of processes in different sectors of industry, such as energy, agriculture, food production, biotechnology, and medicine. The biotechnology sector is improving fast due to a constant technological progress. In particular, the CRISPR/Cas9 technology is probably the biggest revolution in (molecular) biology since PCR (Ledford, 2015). Briefly, CRISPR/Cas9 protects bacteria from invasive genetic elements such as plasmids and viruses (Marraffini, 2015). Taking advantage of this kind of acquired immune system from prokaryotes, a remarkably powerful tool for genome editing based on the CRISPR/Cas9 systems has been developed (Jinek et al., 2012).

The CRISPR/Cas9 system consists of a guide RNA (gRNA) and the endonuclease Cas9 both forming a complex in a way that directs the enzyme to the target site that is complementary to the gRNA promoting site-specific cleavage (Sander and Joung, 2014). The enzymatically generated double strand break (DSB) is being subsequently repaired by the cell through either homologous recombination (HR) or non-homologous end joining (NHEJ) (Iyama and Wilson III, 2012). However, NHEJ works poorly in E. coli (Shuman and Glickman, 2007; Wright et al., 2016) and thus genome editing protocols cannot rely on such a mechanism for this Gram-negative bacterium. Therefore, most approaches aiming to modify chromosomal DNA in E. coli exploit the phage recombinase-mediated homologous recombination (recombineering) using either the Rac prophage system (Zhang et al., 1998; Datta et al., 2006) or the three bacteriophage λ Red proteins Exo, Beta, and Gam (Murphy, 1998; Muyrers et al., 1999; Ellis et al., 2001). In fact, the presence of the λ Red machinery considerably increases mutagenesis efficiency as demonstrated in a recent study (Pyne et al., 2015), especially when combined with the CRISPR/Cas9 technology (Jiang et al., 2013).

Our laboratory established a robust, simple and rapid protocol for multiple, stepwise gene knockout in E. coli using the CRISPR/Cas9 technology in combination with the λ Red machinery (Zerbini et al., 2017). Here, we want to provide our genome editing protocol in detail, which results in high mutagenesis efficiencies and has the potential to be applied in high-throughput approaches.

Materials and Reagents

Pipette tips: GPR-10G (Mettler-Toledo, Rainin, catalog number: 17001862 ), GPR-250 (Mettler-Toledo, Rainin, catalog number: 17001861 ), GPR-1000 (Mettler-Toledo, Rainin, catalog number: 17001859 )
50 ml tube (Corning, Falcon^®, catalog number: 352070 )
Primo^®Vacuum Filter Systems, 250 ml, 0.22 μm, PES (Euroclone, catalog number: EPVPE22250 )
Syringe filters PES 0.22 µm filters (Euroclone, catalog number: EPSPE2230 )
Petri dishes (Corning, Falcon^®, catalog number: 351007 )
MAX efficiency^TM DH5α^TM competent cells (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 18258012 )
Note: You can also use homemade competent cells of E. coli DH5α (see Step E1).
pCasRed plasmid (Zerbini et al., 2017), modified pCas9 plasmid from Addgene (Addgene, catalog number: 42876 ) available upon request
pCRISPR-SacB plasmid (Zerbini et al., 2017), modified pCRISPR plasmid from Addgene (Addgene, catalog number: 42875 ), available upon request
T4 Polynucleotide Kinase (PNK) (New England Biolabs, catalog number: M0201S )
T4 Polynucleotide Kinase (PNK) Reaction Buffer (New England Biolabs, catalog number: B0201S )
Oligonucleotide 1 or gDNA forward (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B)
Oligonucleotide 2 or gDNA reverse (Sigma-Aldrich; desalted, synthesis scale 0.025 µM) for cloning of the mutagenic pCRISPR-SacB-gDNA (see Procedure B)
T4 ligase (New England Biolabs, catalog number: M0202S )
10x T4 ligase buffer (New England Biolabs, catalog number: B0202S )
1 M NaCl (Sigma-Aldrich, catalog number: S7653-1KG )
BsaI enzyme (New England Biolabs, catalog number: R0535L )
DpnI enzyme (New England Biolabs, catalog number: R0176S )
Alkaline phosphatase, Calf Intestinal (CIP, New England Biolabs, catalog number: M0290S )
Wizard^®SV Gel and PCR Clean-Up System (Promega, catalog number: A9282 )
QIAprep Spin Miniprep Kit (QIAGEN, catalog number: 27106 )
ATLAS ClearSight DNA Stain (BioAtlas, catalog number: BH40501 )
Magnesium chloride (MgCl₂) (Sigma-Aldrich, catalog number: M8266-1KG )
Note: See Recipes for preparation of the 100 mM working solution.
Calcium chloride dihydrate (CaCl₂·2H₂O) (Sigma-Aldrich, catalog number: C3306-250G )
Note: See Recipes for preparation of the 100 mM working solution in 15% glycerol.
GoTaq Green Master Mix, 2x (Promega, catalog number: M7123 )
LB broth Miller (Sigma-Aldrich, catalog number: L3522-1KG )
Note: See Recipes for preparation of LB medium.
LB agar (Sigma-Aldrich, catalog number: L2897-1KG )
Note: See Recipes for preparation of Petri dishes with LB-agar.
Kanamycin (Thermo Fisher Scientific, catalog number: 11815024 )
Note: Prepare a stock in a concentration of 50 mg/ml (see Recipes).
Chloramphenicol (Sigma-Aldrich, catalog number: C0378-25G )
Note: Prepare a stock in a concentration of 25 mg/ml (see Recipes).
L-(+)-Arabinose (Sigma-Aldrich, catalog number: A3256-100G )
Note: Prepare a stock in a concentration of 20% L-(+)-arabinose (see Recipes).
Sucrose (Sigma-Aldrich, catalog number: S5016-2.5KG )
Note: Prepare a stock in a concentration of 50% sucrose (see Recipes).
> 99% Glycerol (Acros Organics, catalog number: 158920010 )
Note: Prepare a 75% stock (see Recipes).
LB medium (liquid) (see Recipes)
LB-agar (for Petri dishes) (see Recipes)
Antibiotics (see Recipes)
20% L-(+)-arabinose (see Recipes)
50% sucrose (see Recipes)
75% glycerol solutions (see Recipes)
Sterile 100 mM MgCl₂ (see Recipes)
Sterile 100 mM CaCl₂in 15% glycerol (see Recipes)

Equipment

Pipettes: Pipetman P2L metal ejector (Gilson, catalog number: FA10001M ), Pipetman P10L metal ejector (Gilson, catalog number: FA10002M ), Pipetman P20L metal ejector (Gilson, catalog number: FA10003M ), Pipetman P200L metal ejector (Gilson, catalog number: FA10005M ), Pipetman P1000L metal ejector (Gilson, catalog number: FA10006M )
pH meter (e.g., Fisher Scientific, model: Fisher Scientific^TM accumet^TM AB150, catalog number: 13-636-AB150 )
-80 °C freezer for storage of strains (any commercially available freezer is suitable)
-20 °C freezer (any commercially available freezer is suitable)
Autoclave (e.g., Tuttnauer, model: 5075 EL , 160 L)
Centrifuge, low temperature, up to 6,000 rpm (e.g., Thermo Fisher Scientific, Thermo Scientific^TM, model: Sorvall^TM ST16 )
DNA sequencer (or DNA sequencing service EUROFINS)
Gene Pulser^®/MicroPulser^TM Electroporation Cuvettes, 0.1 cm gap (Bio-Rad Laboratories, catalog number: 1652089 )
MicroPulser^TM Electroporator (Bio-Rad Laboratories, catalog number: 1652100 )
Incubator, temperature scale at least 30-37 °C, shaking range 0-300 r.p.m. (e.g., Eppendorf, New Brunswick^TM, model: Innova^®44 )
MilliQ water system (e.g., Elga Veolia, model: PURELAB^® flex 3 )
Spectrophotometer (e.g., Biochrom, model: WPA CO7500 Colorimeter )
Thermocell mixing block (e.g., BionovaTec, model: MB 102 )
Thermocycler for PCR (e.g., Eppendorf, model: Mastercycler^® nexus )
Transilluminator documentation system (e.g., UviTEC Cambridge, Laboratorium Life Science, model: UVIdoc HD5 )

Procedure

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König, E., Zerbini, F., Zanella, I., Fraccascia, D. and Grandi, G. (2018). Multiple Stepwise Gene Knockout Using CRISPR/Cas9 in Escherichia coli. Bio-protocol 8(2): e2688. DOI: 10.21769/BioProtoc.2688.