Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing

Manjunatha R. Benakanakere, Livia S. Finoti, Urara Tanaka, Gregory R. Grant, Raquel M. Scarel-Caminaga, Denis F. Kinane

Research output: Contribution to journalArticle

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Abstract

Interleukin-8 (IL-8) gene polymorphisms have been considered as susceptibility factors in periodontal disease. However, the functional roles of IL-8 gene haplotypes have not been investigated. Here, we demonstrate for the first time the use of the CRISPR/Cas9 system to engineer the IL-8 gene, and tested the functionality of different haplotypes. Two sgRNAs vectors targeting the IL-8 gene and the naked homologous repair DNA carrying different haplotypes were used to successfully generate HEK293T cells carrying the AT genotype at the first SNP-rs4073 (alias-251), TT genotype at the second SNP-rs2227307 (alias +396), TC or CC genotypes at the third SNP-rs2227306 (alias +781) at the IL-8 locus. When stimulated with Poly I:C, ATC/TTC haplotype, cells significantly up-regulated the IL-8 at both transcriptional and translational levels. To test whether ATC/TTC haplotype is functional, we used a trans-well assay to measure the transmigration of primary neutrophils incubated with supernatants from the Poly I:C stimulation experiment. ATC/TTC haplotype cells significantly increased transmigration of neutrophils confirming the functional role for this IL-8 haplotype. Taken together, our data provides evidence that carriage of the ATC/TTC haplotype in itself may increase the influx of neutrophils in inflammatory lesions and influence disease susceptibility.

Original languageEnglish
Article number31180
JournalScientific reports
Volume6
DOIs
Publication statusPublished - Aug 8 2016

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Clustered Regularly Interspaced Short Palindromic Repeats
Interleukin-8
Haplotypes
Genes
Single Nucleotide Polymorphism
Poly I-C
Neutrophils
Genotype
Gene Editing
Disease Susceptibility
Periodontal Diseases
DNA Repair

All Science Journal Classification (ASJC) codes

  • General

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Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. / Benakanakere, Manjunatha R.; Finoti, Livia S.; Tanaka, Urara; Grant, Gregory R.; Scarel-Caminaga, Raquel M.; Kinane, Denis F.

In: Scientific reports, Vol. 6, 31180, 08.08.2016.

Research output: Contribution to journalArticle

Benakanakere, Manjunatha R. ; Finoti, Livia S. ; Tanaka, Urara ; Grant, Gregory R. ; Scarel-Caminaga, Raquel M. ; Kinane, Denis F. / Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing. In: Scientific reports. 2016 ; Vol. 6.
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