Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCB089

Poster Communications

CRISPR/Cas-mediated targeting and visualization of long non-coding RNAs in human endothelial cells

S. Seredinski1,2, M. S. Leisegang1,2, R. Brandes1,2

1. Vascular Research Centre Frankfurt am Main, Frankfurt am Main, Hessen, Germany. 2. German Center of Cardiovascular Research (DZHK), Partner site RheinMain,, Frankfurt, Germany.

The CRISPR-Cas system is a constantly expanding toolbox for editing the genomic landscape of cells. So far, applications of CRISPR-Cas9 are limited to DNA. Targeting of RNA was mostly neglected although RNA plays a crucial role in every cellular process. The recently published CRISPR/Cas13a from Leptotrichia wadei (LwaCas13a) exclusively targets and cleaves RNA with the help of a matching guide RNA. Here we used the newly developed catalytically inactive, msfGFP-labeled LwadeadCas13a to track down and visualize lncRNAs such as LISPR1 (long intergenic noncoding RNA antisense to S1PR1), NEAT1 and HIF1α-AS1. We were able to show that LISPR1 is located in the nucleus as well as the cytoplasm which confirms previous Western Blot analysis. In contrast NEAT1 and HIF1α-AS1 mainly seem to be accumulated in nuclear speckles. We addressed and optimized the transfection efficiency in HEK cells and human umbilical vein endothelial cells by fluorescence assisted cell sorting (FACS). With immunoprecipitation of LwadCas13-msfGFP we were able to specifically enrich lncRNAs such as LISPR1. Taken together, CRISPR/Cas13 is a new and powerful tool to target RNA. It will help to elucidate mechanisms of regulation of gene expression by RNA.

Where applicable, experiments conform with Society ethical requirements