title: Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing.
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.
authors: Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Manjunatha R
authors: MR
authors: Benakanakere
authors: Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Oral Diagnosis and Surgery, School of Dentistry at Araraquara, UNESP- Univ Estadual Paulista, SP, Brazil.
authors: Livia S
authors: LS
authors: Finoti
authors: Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Urara
authors: U
authors: Tanaka
authors: Department of Genetics, Perelman School of Medicine, University of Pennsylvania, PA, USA.
authors: Gregory R
authors: GR
authors: Grant
authors: Department of Morphology, School of Dentistry at Araraquara, UNESP- Univ Estadual Paulista, SP, Brazil.
authors: Raquel M
authors: RM
authors: Scarel-Caminaga
authors: Department of Periodontics, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.|Department of Pathology, School of Dental Medicine, University of Pennsylvania, Philadelphia, PA, USA.
authors: Denis F
authors: DF
authors: Kinane
mesh_terms: D064113:CRISPR-Cas Systems; D000072669:Gene Editing; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics; D009504:Neutrophils / Q000378:metabolism*; D011070:Poly I-C / Q000494:pharmacology; D020641:Polymorphism, Single Nucleotide; D014176:Protein Biosynthesis / Q000187:drug effects / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects / Q000235:genetics*
chemical_list: C508606:CXCL8 protein, human; D016209:Interleukin-8; D011070:Poly I-C