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PLoS Genet. 2017 Aug 21;13(8):e1006961. doi: 10.1371/journal.pgen.1006961. eCollection 2017 Aug.

Positional cloning of quantitative trait nucleotides for blood pressure and cardiac QT-interval by targeted CRISPR/Cas9 editing of a novel long non-coding RNA.

Author information

1
Program in Physiological Genomics, Center for Hypertension and Personalized Medicine, Department of Physiology and Pharmacology, University of Toledo College of Medicine and Life Sciences, Toledo, OH, United States of America.
2
Department of Radiology, University of Toledo Medical Center, Toledo, OH, United States of America.
3
Department of Cell & Molecular Biology, Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN, United States of America.

Abstract

Multiple GWAS studies have reported strong association of cardiac QT-interval to a region on HSA17. Interestingly, a rat locus homologous to this region is also linked to QT-intervals. The high resolution positional mapping study located the rat QT-interval locus to a <42.5kb region on RNO10. This region contained no variants in protein-coding sequences, but a prominent contiguous 19bp indel polymorphism was noted within a novel predicted long non-coding RNA (lncRNA), which we named as Rffl-lnc1. To assess the candidacy of this novel lncRNA on QT-interval, targeted CRISPR/Cas9 based genome-engineering approaches were applied on the rat strains used to map this locus. Targeted disruption of the rat Rffl-lnc1 locus caused aberrant, short QT-intervals and elevated blood pressure. Further, to specifically examine the significance of the 19bp polymorphism within the Rffl-lnc1 locus, a CRISPR/Cas9 based targeted knock-in rescue model was constructed by inserting the 19bp into the strain which contained the deletion polymorphism. The knock-in alleles successfully rescued the aberrant QT-interval and blood pressure phenotypes. Further studies revealed that the 19bp polymorphism was necessary and sufficient to recapitulate the phenotypic effect of the previously mapped <42.5kb rat locus. To our knowledge, this study is the first demonstration of a combination of both CRISPR/Cas9 based targeted disruption as well as CRISPR/Cas9 based targeted knock-in rescue approaches applied for a mammalian positional cloning study, which defines the quantitative trait nucleotides (QTNs) within a rat long non-coding RNA as being important for the pleiotropic regulation of both cardiac QT-intervals and blood pressure.

PMID:
28827789
PMCID:
PMC5578691
DOI:
10.1371/journal.pgen.1006961
[Indexed for MEDLINE]
Free PMC Article

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