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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

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.
Department of Radiology, University of Toledo Medical Center, Toledo, OH, United States of America.
Department of Cell & Molecular Biology, Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN, United States of America.


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.

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