title: CRISPR-based genome editing of clinically important Escherichia coli SE15 isolated from indwelling urinary catheters of patients.
abstract: Urinary tract infections (UTIs) are clinically important problems that lead to serious morbidity and mortality, and indwelling urinary catheters are a major factor of UTIs. In this study, we applied clustered regularly interspaced short palindromic repeats (CRISPR) genome editing to generate ΔluxS mutant strains from clinical isolates of Escherichiacoli SE15, which is one of major pathogens and can cause colonization and biofilm formation in the catheter. A major regulatory pathway of such biofilm formation on medical devices is the quorum sensing mechanism via small molecule autoinducer-2 synthesized by LuxS enzyme. Here, we used the CRISPR-Cas9 system for precise deletion of luxS gene in clinical isolate E. coli SE15. To this end, we constructed a donor DNA for homologous recombination to delete 93 bases in the chromosomal target (luxS) and observed the success rate of luxS deletion to be 22.7 %. We conducted biofilm assay to observe decreased biofilm formation in the E. coil SE15 ΔluxS mutants compared to wild-type E. coil SE15. Quantitative real-time PCR analysis of E. coil SE15 ΔluxS mutants showed that the expression of luxS was below detection level. We also observed that the relative mRNA levels of biofilm-formation-related genes, such as mqsR, pgaBC and csgEF, were significantly decreased in E. coil SE15 ΔluxS mutants compared to wild-type. We conclude that genome editing by CRISPR-Cas9 system is an effective tool to dissect the molecular mechanism of biofilm formation in medically important strains, and the study may serve as a basis for developing novel medical intervention against UTIs caused by biofilm.
authors.affiliation: Department of Biological Engineering Kyonggi University, 154-42 Gwanggyosan-ro Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea.
authors.forename: Sangrim
authors.initials: S
authors.lastname: Kang
authors.affiliation: Department of Life Science Kyonggi University, 154-42 Gwanggyosan-ro Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea.
authors.forename: Jinsoo
authors.initials: J
authors.lastname: Kim
authors.affiliation: Stem Cell Institute, Korea University, 145 Anam-ro, Seongbuk-gu, Seoul, Republic of Korea, 154-42 Gwanggyosan-ro Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea.
authors.forename: Junho K
authors.initials: JK
authors.lastname: Hur
authors.affiliation: Department of Biological Engineering Kyonggi University, 154-42 Gwanggyosan-ro Yeongtong-gu, Suwon-si, Gyeonggi-do, Republic of Korea.
authors.forename: Sang-Seob
authors.initials: SS
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D018441:Biofilms; D064113:CRISPR-Cas Systems; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D002408:Catheters, Indwelling / Q000382:microbiology; D002876:Chromosomes, Bacterial / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D017353:Gene Deletion; D000072669:Gene Editing; D015964:Gene Expression Regulation, Bacterial; D006801:Humans; D053038:Quorum Sensing; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D062885:Urinary Catheters / Q000382:microbiology*
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D012333:RNA, Messenger; D013437:Carbon-Sulfur Lyases; C117753:LuxS protein, Bacteria