Identification of RBM20 splicing kinases and base editing of cardiomyopathy-associated RBM20 p.P638L mutation

Background/Aims
RBM20 encodes the RNA binding motif protein 20, which acts as a cardiac splicing factor. Mutations in RBM20 are associated with clinically severe forms of cardiomyopathy. Most of the known pathogenic RBM20-mutations are localized in the highly conserved arginine- and serine-rich (RS) domain of the protein. These mutations lead to a mislocalization of the protein from the nucleus to the cytoplasm which is presumably caused by a misphosphorylation of the RS domain. SR-protein specific kinases (SRPKs) phosphorylate the RS domain of SR proteins. The phosphorylation of the RS domain has an important role for the subcellular localization and the protein-protein interactions of SR proteins. It is not completely understood which SRPKs are responsible for the phosphorylation of RBM20. One aim of this project was to identify kinases involved in phosphorylation of RBM20. Additionally, we aimed to establish and apply base editing to correct a pathogenic RBM20 mutation within the RS domain as base editing is a potent gene editing tool even in postmitotic cells like cardiomyocytes.

Methods/Results
Using CRISPR/Cas9 mediated gene editing we generated knock-outs (k.o.) of SRPK1 and SRPK2 in HEK-293 cells and k.o. of SRPK1 and SRPK3 in induced pluripotent stem cells (iPSC). Interestingly, a k.o. of SRPK2 seemed to be lethal in iPSCs. This suggests that SRPK2 is an essential kinase involved in the survival of these cells. Subcellular RBM20 localization was neither disturbed in SRPK1-k.o. and SRPK2-k.o. HEK-293 cells nor in SRPK1-k.o. iPSC-derived cardiomyocytes (iPSC-CMs). Unfortunately, in contrast to adult human cardiomyocytes SRPK3 was not expressed in iPSC-CMs so that the effect of SRPK3-k.o. could not be analyzed.

Using CRISPR/Cas9 combined with homology directed repair we generated an iPSC line which is heterozygous for the pathogenic RBM20 p.P638L mutation. In this line the mislocalization of the mutated RBM20 to the cytoplasm could be confirmed via immunofluorescence microscopy. This cell line was then used for adenine base editing. First, we constructed a vector which carries both the base editor and the promotor/scaffold for guide RNA expression. This construct enhances a successful editing of the respective cells as only a single plasmid hat to enter the cells. This plasmid was electroporated into the iPSC-p.P638L line and the efficiency was about 65% as confirmed with fluorescence-assisted cell sorting. Using Sanger- and Amplicon-sequencing we observed a successful editing of the desired base. Unfortunately, another adenine also localized within the editing window and resulted in an unwanted missense editing event. The consequential amino acid exchange also leads to a mislocalization of RBM20.

Discussion/Outlook
As no RBM20 mislocalization was observed after SRPK k.o. it might be suggested that either none of the analyzed SRPKs is responsible for RBM20 phosphorylation or that a single knockout is not sufficient to affect the localization of the protein. Currently we are working on the direct analysis of RBM20 phosphorylation using Phos-Tag-Gels. Additionally, we will apply an SRPK1/2-inhibitor to test the effect of a dose dependent simultaneous inhibition of both kinases on RBM20.

For base editing we are now working with a recently published nearly PAM-less base editor. This editor enables a more flexible choice of the guide RNA and the editing window and might enable an appropriate editing without unwanted side effects.