Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9
© The Author(s) 2016
Received: 1 October 2016
Accepted: 25 November 2016
Published: 1 December 2016
Microbial genome editing is a powerful tool to modify chromosome in way of deletion, insertion or replacement, which is one of the most important techniques in metabolic engineering research. The emergence of CRISPR/Cas9 technique inspires various genomic editing methods.
In this research, the goal of development of a fast and easy method for Escherichia coli genome editing with high efficiency is pursued. For this purpose, we designed modular plasmid assembly strategy, compared effects of different length of homologous arms for recombination, and tested different sets of recombinases. The final technique we developed only requires one plasmid construction and one transformation of practice to edit a genomic locus with 3 days and minimal lab work. In addition, the single temperature sensitive plasmid is easy to eliminate for another round of editing. Especially, process of the modularized editing plasmid construction only takes 4 h.
In this study, we developed a fast and easy genome editing procedure based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci.
Microbial genome editing is for site-specific chromosome modification, which is one of the most useful techniques in research and plays an important role in metabolic engineering and molecular biology research. Current methods include homologous recombination-based gene targeting [1–3], zinc finger nucleases (ZFNs)  and transcription activator-like effector nucleases (TALEN) [5, 6]. However, these techniques are often inefficient, time consuming, laborious, or expensive to use. Thus, a rapid and simple method capable of performing precise genome editing with minimal cost remains to be desired.
The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system is an RNA-guided immune system in many bacteria . This system is able to recognize and generate double-strand breaks (DSBs) at target sequence. Following the DSB event, native DNA repair will be initiated through non-homologous end joining or homologous recombination in the presence of an exogenous homologous donor sequence . The only restriction for designing a CRISPR/Cas9 guiding sequence is need for a protospacer adjacent motif (PAM) close to genomic target site . The typeII CRISPR/Cas9 system of Streptococcus pyogenes, which requires a mature CRISPR RNA (crRNA), a trans-activating crRNA (tracrRNA) and a DNA endonuclease Cas9 has attracted significant attention and been harnessed for targeted genome editing in different organism, both Eukaryotes and Prokaryotes, such as E. coli [10–12], human , mice  and zebrafish . Thus far, the CRISPR-Cas9 system is a powerful and revolutionary tool for genome editing.
The CRISPR/Cas9 system has been widely used for eukaryotic genomes but its applications in bacterial genomes were less studied. CRISPR/Cas9 system was firstly exploited for bacterial genomic editing by Jiang et al., the work was a huge breakthrough for microbial genomic editing, however, high editing efficiency in E. coli was not achieved . Recently, researchers achieved very good results in development of Cas9 based gnomic editing techniques [10–12, 16]. While these methods improved the genome editing techniques, some aspects could be further improved, such as editing efficiency, lab hours, convenience of usage and standard protocols. To tackle these problems, we started the work of development of a simple and fast method for bacterial genome editing with high efficiency. For this purpose, multi-plasmid system was experimented and found to be relatively laborious and time consuming. Low editing efficiency was observed with the plasmid/linear DNA system, especially with wild type and E. coli cell factory strains. This was probably due to their strong restriction systems which degrade the linear donor DNAs before integration into chromosome . To avoid these drawbacks, our strategy was designed to edit genome with only one plasmid, which carried all functional parts to make the technique very easy and fast to practice and achieve high efficiency.
Escherichia coli MG1655 and DH 5 α were grown at 30 °C in lysogeny broth (LB, 1% (w/v) tryptone, 0.5% (w/v) yeast extract, 1% (w/v) NaCl). Kanamycin (50 mg/L for E. coli) and Ampicillin (100 mg/L for E. coli) were added to the medium as appropriate. 1% (w/v) glucose was added to the culture for Cas9 repression and l-arabinose (2 g/L final concentration) was used for Cas9 induction when necessary. IPTG and X-gal for blue/white selection were added at concentrations of 0.1 mM and 40 µg/mL, respectively .
Escherichia coli MG1655 were grown in 5 mL LB cultures at 30 °C to an OD600 of 0.6 and then made electrocompetent by concentrating 100-fold and washing three times with ice-cold 10% glycerol. 10–100 ng of PCR target plasmid was used for electroporation, shocked cells were added to 1 mL LB, incubated 1 h at 30 °C, and then plated on LB petri dishes carrying appropriate antibiotics.
The Cas9 gene from Cas9 expression plasmid (Addgene reference number: 42876), gRNA without N20 (5′- GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTT-3′) and its promoter (5′- CTAGGTTTATACATAGGCGAGTACTCTGTTATGGAGTCAGATCT-3′) which were all directly synthetized. pKD46 modular (contains: exo, bet, gam, rep and arabinose operon) was used for plasmid construction as backbone.
pCas9 series and pRed_Cas9 series other than pRed_Cas9_recA_Δpoxb41 and pRed_Cas9_recA_ΔlacZ41 were assembled with the Gibson method, of which DNA oligo primers were designed with j5 and Device Editor . DNA templates were PCR-amplified with Phusion polymerase (NEB) or directly synthesized. PCR products were gel purified, digested with DpnI before Gibson amplification.
For modular design, the pRed_Cas9_recA_Δpoxb300 plasmid was used as template for the PCR amplification of target modules. Homologous arms of H1 and H2 were obtained from genomic DNA of E. coli MG1655. pRed_Cas9_recA_Δpoxb41, pRed_Cas9_recA_Δpoxb::rfp41 and pRed_Cas9_recA_ΔlacZ41 were assembled using the modular assembly method.
Strain and plasmid availability
The plasmids used for this work
Exo, bet, gam, rep and arabinose operon
Arabinose operon, Cas9, gRNA with N20 and with homologous arms of about 300 bp for poxb
Derived from pKD46, exo, bet, gam, arabinose operon, Cas9, gRNA with N20 and with homologous arms of about 300 bp for poxb deletion
Derived from pRed_Cas9_ΔpoxB300, and with homologous arms of about 300 bp with rfp replace poxb
Derived from pRed_Cas9_ΔpoxB300, and with homologous arms of about 100 bp for poxb deletion
Derived from pRed_Cas9_ΔpoxB300, and with homologous arms of about 50 bp for poxb deletion
Derived from pRed_Cas9_ΔpoxB300, recA
Derived from Red_Cas9_recA_Δpoxb300, and with homologous arms of about 41 bp for poxb deletion
Derived from Red_Cas9_recA_Δpoxb300, and with homologous arms of about 41 bp for rfp replace poxb
Derived from Red_Cas9_recA_Δpoxb300, and with homologous arms of about 41 bp for lacZ deletion
Genome editing procedure
A pair of 24 nt oligos and a pair of 86 nt oligos were designed to target genomic locus for editing. A simple DNA annealing procedure was used to obtain the N20 part and donor DNA part, which were assembled with Part1 and Part2 with a Golden Gate assembly reaction to the editing plasmid. Then the plasmid was transformed into E. coli, followed by plating on LB+ Kan+ 1% (w/v) glucose plate, and culturing at 30 °C. The resulting strain was grown in LB+ Kan for 2 h before 2 g/L l-arabinose induction for Cas9 and gRNA expression. After culturing of 6 h, the culture was plated on LB+ Kan+ l-arabinose agar plates, and the colonies were analyzed by colony PCR with a forward primer upstream of the left homology arm and a reverse primer downstream of the right homology arm. Colonies with expected PCR product were subjected to verification by DNA sequencing for further confirmation. At last, the temperature sensitive editing plasmid is cured by growing the edited strains at 37 °C overnight. A detailed genome editing protocol is provided in supplemental file (Additional file 1).
Editing efficiency calculation
For editing efficiency calculation, cell culture after induction process was diluted 100 fold, of which 20 μL was plated on LB dishes for proximate colony counts. Colonies was screened by PCR and DNA sequencing to indentify correctly edited clones. With each experiment, 48 colonies are screened by colony PCR and the correct colonies were used for further verification by DNA sequencing. The editing efficiency equals the percentage of the correct colonies in all analyzed colonies. All experiments were repeated three times to obtain the means and standard variations.
When lacZ gene was targeted, cells were plated on IPTG and X-gal plates. The apparent editing efficiency was ratio of white colonies of total colonies. For further verification, white colonies were identified by colony PCR and DNA sequencing to obtain the real editing efficiency, which equaled ratio of the correctly edited colonies of total colonies.
CRISPR/Cas9 based chromosomal gene deletion of poxB with one plasmid construction and one transformation
A non-essential gene poxB(GeneID: 946132) was selected as a target gene in E. coli MG1655 for genome editing . An editing plasmid, pAra_Cas9_Δpoxb300, was constructed to delete 513 bp chromosomal fragment within poxB, which employed 300 bp homologous arms to recombine with the target locus. The plasmid was electroporated into E. coli MG1655 to initiate genome editing. After culturing and induction with l-arabinose (Fig. 1), the culture was diluted 100 fold, of which 20 μL were plated to obtain edited strains. A few hundred colonies were obtained. For the control experiment, cells were cultured without l-arabinose induction, thus, little Cas9 was expressed. The control group had many more colonies appeared on plate to form a bacterial meadow. This result demonstrated that Cas9 and gRNA were successfully expressed and functional. After arabinose induction, gRNA guided Cas9 to have killed most bacterial population that did not undergo poxB editing. By colony PCR and DNA sequencing, 20.8 ± 8.8% random picked colonies from experiment group were determined to have been successfully edited, while no correctly edited ones from control group. This result demonstrated that our genome editing method was functional as expected.
λRed recombinases improved editing efficiency
The one-plasmid genome editing system was then carried out for chromosomal gene replacement experiment. Plasmid pRed_Cas9_Δpoxb::rfp300 was constructed for replacement of poxb with rfp gene. In this study, 513 bp of poxb was replacement by an 815 bp fragment, which consisted of rfp gene and SD sequence. A high editing rate of 100% was also obtained, part of the agarose gel electrophoresis of colony PCR was shown in Fig. 2b. The results demonstrated that with supplementation of λRed recombinases, the one plasmid strategy worked with 100% efficiency either with genomic knockout or replacement. And the target deletion length did not affect editing efficiency at least within 1500 bp range, which is long enough for normal gene knockout experiments.
Genomic editing efficiency decreased with shorter homologous recombination arms
The editing efficiency and colony number at poxB locus with different length of homologous arms
Homologous arm length
Proximate colony number
297 bp plus 298 bp
100 ± 0%
101 bp plus 101 bp
69.3 ± 1.17%
51 bp plus 51 bp
Addition of recA enabled short homologous recombination arms for successful genomic editing
For our one-plasmid genomic editing system, the most laborious part of work was plasmid construction. If the homologous arms were short enough, the two homologous arms could be assembled by primer embedding, instead of PCR and ligation. To simplify plasmid construction, it was important to make shorter homologs arms work for this method.
Reports have shown that recA improved red/ET recombination efficiency . To increase editing efficiency, recA gene was added to the one plasmid system to create pRed_Cas9_recA_Δpoxb41 plasmid which carried λRed recombinases, CRISPR/Cas9 system, recA and two homologous arms of 41 bp. An editing efficiency of 13.8 ± 7.9% was achieved in E. coli MG1655 with poxB gene deletion. Parts of the agarose gel electrophoresis of colony PCR is shown as Fig. 2c. For gene replacement, pRed_Cas9_recA_Δpoxb::rfp41 was constructed for replacement of poxb with rfp gene. With 41 bp homologous arms, an editing efficiency of 16.7 ± 3.8% was achieved. The editing efficiency with 41 bp homologous arms is acceptable for genome editing experiment, since even with this efficiency, at least one successfully edited strain could be obtained from seven to eight colonies. Furthermore, colony PCR screening is not laborious and is to be performed even with high editing efficiency experiments.
One-plasmid method enabled convenient multi-round genome editing
Efficiency of the two round consecutive editing with one-plasmid method
First round at poxB
13.8 ± 7.9%
Second round at lacZ
89.7 ± 0.9%
19.9 ± 4.1%
Development of modularized assembly strategy for plasmid construction
Modular Part1 and part2 were the basic compositions for plasmid construction, which were ready made for editing of different loci, as showed in Fig. 3b. Part1 consisted of a resistance gene, an arabinose-inducible pBAD promoter with Cas9 and recA, a terminator, and a constitutive promoter for gRNA. Part2 consisted the temperature-sensitive replicon repA101ts, gRNA without N20 leading sequence, exo, bet, gam, rep and araC. N20 parts and donor DNA parts were annealed from synthesized DNA oligos. A pair of 24 nt oligos and a pair of 86 nt oligos were needed for editing of one locus. Only a simple DNA annealing experiment and a Golden Gate assembly experiment were performed for the plasmid construction, without any necessities for PCR, DNA purification, or gel electrophoresis. The construction could be completed within four hours with minimal lab work. The assembled plasmid from these 4 parts was illustrated in Fig. 3c.
The development of genomic DNA editing protocol with one plasmid construction and one transformation
To develop a mature protocol for fellow researchers to use without further try and error, we went through the experiment process several times and optimized parameters to ensure successful genome editing. The protocol was divided in 4 steps. The first and second steps were to construct two modular parts, N20 part and donor DNA part. The third step was to assemble two ready-made modular parts with N20 and donor DNA parts with Golden Gate method in one reaction. The last step included one transformation and one culturing practice to obtain edited strains. The detailed protocol is presented in Additional file 1.
Comparison of different methods using CRISPR/Cas9 system
Plasmid curing time
pRed_Cas9_recA system this study
13.8 ± 7.9%(41 bp homologous arms) 100%(more than300 bp homologous arms)
3 days (including plasmid construction, genome editing and plasmid curing)
System established by Li 
Two plasmid and linear donr DNA
ND (estimated 4 days)
System established by Jiang 
Two plasmid and/or linear donr DNA
4 days(without plasmid construction)
System established by Jiang 
Two plasmid and/or linear donr DNA
65 ± 14%
ND (estimated more than 4 days without plasmid construction)
The method also has an advantage for editing of multiple loci. The system requires only one temperature sensitive plasmid, which makes plasmid curing process easy and fast. In continuous process with our technique, X loci could be edited within 3× days. This rapid and simple method for E. coli genome editing has already been used in our research group, for example editing promoters of crt, idi and dxs in QL002 strain . And it has also already been adopted by other labs in our institute.
High throughput and automatic genomic editing is one highly desired technique by molecular biologists [24, 25]. Due to its modular design, this method could be applied to develop automatic genome editing techniques. In plasmid construction process, DNA annealing and Golden Gate assembly process could be easily transformed to computer controlled practice with a liquid handler platform [26, 27]. Plasmid transformation and induction process could be automated as the MAGE system [24, 28]. Colonies PCR screening process can also be performed by automatic system [26, 27]. We expect a combined automatic system to be able to perform high-throughput, rationally designed genome editing based on our methods.
Colony PCR was used to identify successfully edited clones based on the size of PCR products in this work. However, as shown in Fig. 2c, some colony PCRs turned out to have neither edited band nor original band, just empty lanes. These colony PCRs were repeated with more sets of primers and were confirmed not to be able to obtain amplification products. A newly published article suggested that E. coli strains might possess a distinct end-joining activity that repairs double strand breaks (DSBs) and generate genome rearrangements. This mechanism, named alternative end-joining (A-EJ), is characterized by extensive DNA end restriction and large chromosomal loss . We hypothesized that the colonies without PCR products might be E. coli cells that survived CRISPR mediated DSBs by A-EJ and underwent large DNA deletion. This hypothesis was partially proved by editing of lacZ locus. As illustrated in Table 3, 89% edited strains were white which apparently had lacZ knocked out. However, only 19% was confirmed to have shortened lacZ gel bands by colony PCR, which was the indication of precise editing as designed. Others showed no band or smear on gel, indicating changed chromosomal status around the lacZ locus. To analyze a larger area, primers targeting a region up to 7.5 kb around the Cas9 cleavage locus were designed and applied. However, no target PCR products were obtained with these primers either. Due to cost reason, we had not sequence these strains yet to find out their genomic changes. According to a newly published article, our result suggest CRISPR mediated DSBs may induce A-EJ repair in E. coli, and cause very large genomic deletion or rearrangement .
The method established in this paper has several advantages compared to others, however, its editing efficiency for short homologous arms did not reach 100%. There are some strategies we would exploit to improve in the near future. For example, using new CRISPR proteins, such as Cpf1 might help to improve recombination frequency by its different cleavage pattern . Moreover, we might further improve homologous recombination efficiency by experimenting with more recombinases from various sources [32, 33].
In this study, we achieved the goal of development of a very fast and easy genome editing technique with high efficiency based on CRISPR/Cas9 system that only required the work of one plasmid construction and one transformation, which allowed modification of a chromosome locus within 3 days and could be performed continuously for multiple loci.
CB, XZ and DZ conceived the experiments. DZ, SY, BX, HS, LY and JL performed the experiments. CB and DZ analyzed the results. CB and DZ wrote the paper. All authors read and approved the final manuscript.
This research was financially supported by the National High Technology Research and Development Program of China (2015AA020202), Tianjin Key Technology R&D program of Tianjin Municipal Science and Technology Commission (Y5M2121111) and Novo Nordisk—Chinese Academy of Sciences (NN-CAS) Research Fund (NNCAS-2015-2).
The authors declare that they have no competing interests.
Availability of data and materials
This research was supported by the National High Technology Research and Development Program of China (2015AA020202), Tianjin Research Program of Application Foundation and Advanced Technology (15JCYBJC24200) and Novo Nordisk-Chinese Academy of Sciences (NN-CAS) Research Fund (NNCAS-2015-2).
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