A CRISPR/Cas9 System Based Targeted Disruption of a Novel Rat Long Non‐coding RNA in a Rat Genetic Model of Hypertension

Abstract

High resolution mapping of a human cardiovascular locus (QT-interval) in the homologous genomic segment spanning <42.5 kb on rat chromosome 10 was previously reported as a validation study. The critical genomic segment contains a single protein-coding gene, rififylin (Rffl) without any exonic variants. Both mRNA and protein levels of Rffl were altered between the strains (Dahl S and Lewis) used for mapping. A functional study points to altered rate of endocytic recycling as the underlying mechanism, through which Rffl operates to control both cardiac QT-intervals and hypertension. Despite these important findings, the identity of the quantitative trait nucleotides that regulates the expression of Rffl is unknown. By comparative mapping of human, mouse and rat genomes, a novel long non-coding RNA (lncRNA), which we refer to as Rffl-lnc1, was predicted to target Rffl. Interestingly, compared to the S rat, the Lewis alleles of Rffl-lnc1 are polymorphic, with a large 19bp deletion. To further assess the role of Rffl-lnc1, the CRISPR/Cas9 system was applied to construct a 19bp mutant model of the S rat. CRISPR/Cas9 reagents were generated and verified in rat C6 cells. Single cell embryos from the S rat were microinjected and transferred into surrogate females. Out of 67 pups born, 21 were mutants at the targeted 19bp locus. To our knowledge, these rats represent the first mutant strains of a long non-coding RNA and serve as novel models for delineating the function of the lncRNA, Rffl-lnc1, in cardiovascular diseases. Research support to BJ: NHLBI/NIH 020176, 112641.