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Sci Rep. 2017 Dec 6;7(1):17077. doi: 10.1038/s41598-017-17387-x.

CRISPR/Cas9 mediated mutation of mouse IL-1α nuclear localisation sequence abolishes expression.

Author information

1
Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK.
2
Transgenic unit, Faculty of Biology, Medicine and Health, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK.
3
Division of Neuroscience and Experimental Psychology, School of Biological Sciences, Faculty of Biology, Medicine and Health, Manchester Academic Health Science Centre, University of Manchester, AV Hill Building, Oxford Road, Manchester, M13 9PT, UK. david.brough@manchester.ac.uk.

Abstract

Inflammation is a host defense process against infection. Inflammatory mediators include cytokines of the interleukin-1 family, such as IL-1α and IL-1β. Unlike IL-1β, IL-1α carries an N-terminal nuclear localisation sequence (NLS) and is trafficked to the nucleus. The importance of IL-1α nuclear localisation is poorly understood. Here, we used CRISPR/Cas9 to make inactivating mutations to the NLS on the Il1a gene. A colony of NLS mutant mice was successfully generated with precise knock-in mutations to incapacitate NLS function. NLS mutant mice had no gross changes in immunophenotype or inflammatory response but, surprisingly, failed to express IL-1α. We deduced that, in making specific mutations in the Il1a gene, we also mutated a long-noncoding (lnc)RNA in the complementary strand which has cis-regulatory transcriptional control of the Il1a gene itself. The mutations generated in the Il1a gene also result in mutation of the lncRNA sequence and a predicted alteration of its secondary structure, potentially explaining a subsequent failure to function as a transcriptional activator of Il1a expression. Thus, lncRNA secondary structure may regulate IL-1α expression. Our results serve as a cautionary note that CRISPR -mediated genome editing without full knowledge of genomic context can result in unexpected, yet potentially informative observations.

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