TY - JOUR
T1 - Investigation of the functional role of human Interleukin-8 gene haplotypes by CRISPR/Cas9 mediated genome editing
AU - Benakanakere, Manjunatha R.
AU - Finoti, Livia S.
AU - Tanaka, Urara
AU - Grant, Gregory R.
AU - Scarel-Caminaga, Raquel M.
AU - Kinane, Denis F.
N1 - Funding Information:
This research was in part supported by DE024160-01A1 from the National Institute of Dental and Craniofacial Research, NIH to DFK. LSF would like to acknowledge the financial supports from Capes/PDSE under Process BEX 2563/14-2, Government of Brazil. Authors would like to thank Next Generation Sequencing Core, Perelman School of Medicine, University of Pennsylvania.
PY - 2016/8/8
Y1 - 2016/8/8
N2 - 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.
AB - 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.
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U2 - 10.1038/srep31180
DO - 10.1038/srep31180
M3 - Article
C2 - 27499075
AN - SCOPUS:84981214270
VL - 6
JO - Scientific Reports
JF - Scientific Reports
SN - 2045-2322
M1 - 31180
ER -