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

Meijia Hou; Simeng Sun; Qizheng Feng; Xiumei Dong; Ping Zhang; Bo Shi; Jiali Liu; Dongfang Shi

doi:10.7717/peerj.8881

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

PeerJ. 2020 Apr 6:8:e8881. doi: 10.7717/peerj.8881. eCollection 2020.

Authors

Meijia Hou¹, Simeng Sun¹, Qizheng Feng¹, Xiumei Dong¹, Ping Zhang¹, Bo Shi¹, Jiali Liu¹, Dongfang Shi¹

Affiliation

¹ Veterinary Medicine, Northeast Agricultural University, Harbin, Heilongjiang Province, China.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 is an emerging gene-editing technology that is widely used in prokaryotes and eukaryotes. It can realize the specific manipulation of the genome efficiently and accurately. CRISPR/Cas9 coupled λ-Red recombination technology was used to perform genome editing in different genes. For finding an efficient method to edit the virulence genes of enterotoxigenic E. coli (ETEC), the two-plasmid system was used. The coding sequence (CDS) region of the estA, eltI, estB, eltIIc1, and faeG locus were deleted. The coding region of estB was substituted with estA. Gene recombination efficiency ranged from 0 to 77.78% when the length of the homology arm was from 50 to 300 bp. Within this range, the longer the homology arm, the higher the efficiency of genetic recombination. The results showed that this system can target virulence genes located in plasmids and on chromosomes of ETEC strains. A single base mutation was performed by two-step gene fragment replacement. This study lays the foundation for research on virulence factors and genetic engineering of vaccines for ETEC.

Keywords: CDS; CRISPR-Cas9; ETEC; Single base mutation; Virulence genes.

Grants and funding

Financial support for this study was provided by grants from the National Science and Technology Support Project (2012BAD12B03-3, 2012BAD12B05-2) and the Science and Technology Planning Project of Heilongjiang Province (GC12B303). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.