Genetic editing of the virulence gene of<i> Escherichia coli</i> using the CRISPR system

Hou, M Z; Sun, Simeng; Feng, Qizheng; Dong, Xueyi; Zhang, Ping; Shi, Bo; Liu, Jiali; Shi, Dongfang

doi:10.7717/peerj.8881

Cited by 11 publications

(10 citation statements)

References 26 publications

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“…Organisms of interest can now be labeled using specific-site knock-in and the integrated expression of exogenous fluorescent proteins in hosts based on CRISPR/Cas9. In the case of the ETEC infection, the underlying mechanism can be better understood by various gene modifications, including overexpression, gene deletion, or the precise regulation of virulence factors, to aid in uncovering their functions [35]. A CRISPR-Cas9 system-based continual genome editing strategy, including gene insertions and knock-outs of both single and multiple (up to three) targets in the E. coli genome, has been developed and is now widely applied [36,37].…”

Section: Introductionmentioning

confidence: 99%

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

Tao

et al. 2022

IJMS

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Some prevention strategies, including vaccines and antibiotic alternatives, have been developed to reduce enterotoxigenic Escherichia coli proliferation in animal production. In this study, a wild-type strain of BE311 with a virulent heat-stable enterotoxin gene identical to E. coli K99 was isolated for its high potential for gene expression ability. The whole genome of E. coli BE311 was sequenced for gene analyses and editing. Subsequently, the fluorescent gene mCherry was successfully knocked into the genome of E. coli BE311 by CRISPR/Cas9. The E. coli BE311–mCherry strain was precisely quantified through the fluorescence intensity and red colony counting. The inflammatory factors in different intestinal tissues all increased significantly after an E. coli BE311–mCherry challenge in Sprague–Dawley rats (p < 0.05). The heat-stable enterotoxin gene of E. coli BE311 was knocked out, and an attenuated vaccine host E. coli BE311-STKO was constructed. Flow cytometry showed apoptotic cell numbers were lower following a challenge of IPEC-J2 cells with E. coli BE311-STKO than with E. coli BE311. Therefore, the E. coli BE311–mCherry and E. coli BE311-STKO strains that were successfully constructed based on the gene knock-in and knock-out technology could be used as ideal candidates in ETEC challenge models and for the development of attenuated vaccines.

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Section: Introductionmentioning

confidence: 99%

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

Tao

et al. 2022

IJMS

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“…The evaluation of sgRNA off-target probability is based on E. coli K 12, MG 1655, BL 21( DE 3) instead of wild-type E. coli . It might lead to inaccurate evaluation of sgRNA off-target probability (Hou et al 2020 ). So the sequences of wild-type E. coli irp2 gene need to be compared with those of E. coli using the online sgRNA design tool, and 3 sgRNA sequences were designed according to their conserved region sequences.…”

Section: Discussionmentioning

confidence: 99%

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

et al. 2021

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To construct wild-type E. coli irp2 gene deletion strains, CRISPR/Cas9 gene editing technology was used, and the difficulty and key points of gene editing of wild-type strains were analyzed. Based on the resistance of the CRISPR/Cas9 system expression vector, 4 strains of 41 E. coli strains isolated from Saba pigs were selected as the target strains for the deletion of the irp2 gene, which were sensitive to both ampicillin and kanamycin. Then, CRISPR/Cas9 technology was combined with homologous recombination technology to construct recombinant vectors containing Cas9, sgRNA and donor sequences to knock out the irp2 gene. Finally, the absence of the irp2 gene in E. coli was further verified by iron uptake assays, iron carrier production assays and growth curve measurements. The results showed that three of the selected strains showed single base mutations and deletions (Δirp2-1, Δirp2-2 and Δirp2-3). The deletion of the irp2 gene reduced the ability of E. coli to take up iron ions and produce iron carriers, but not affect the growth characteristics of E. coli. It is shown that the CRISPR/Cas9 knock-out system constructed in this study can successfully knock out the irp2 gene of the wild-type E. coli. Our results providing new insights into genome editing in wild-type strains, which enable further functional studies of the irp2 gene in wild-type E. coli.

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“…Transformants were confirmed by colony PCR and sequencing. After pTarget and pCas curing (Hou et al, 2020), deletions of other genes coding other deaminases, transferases, oxidoreductases, membrane proteins (MPs), and other proteins were conducted. The selection of nonessential genes referred to the Keio collection (Baba et al, 2006; Goodall et al, 2018).…”

Section: Methodsmentioning

confidence: 99%

Whole‐cell biosynthesis of cytarabine by an unnecessary protein‐reduced Escherichia coli that coexpresses purine and uracil phosphorylase

Ping

Ruxian

Mengping

et al. 2022

Biotech & Bioengineering

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Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.

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Genetic editing of the virulence gene of Escherichia coli using the CRISPR system

Cited by 11 publications

References 26 publications

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

Whole Genome Sequencing and CRISPR/Cas9 Gene Editing of Enterotoxigenic Escherichia coli BE311 for Fluorescence Labeling and Enterotoxin Analyses

New insights into the construction of wild-type Saba pig-derived Escherichia coli irp2 gene deletion strains

Whole‐cell biosynthesis of cytarabine by an unnecessary protein‐reduced Escherichia coli that coexpresses purine and uracil phosphorylase

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