Investigating CRISPR-CAS13b as a tool for the RNA editing of CFTR mRNA with premature stop codon

Background and Rationale Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. Mutant CFTR gene coding for transcripts with premature termination codons (PTCs) is responsible for truncated CFTR protein and for a severe form of the disease. In a precision medicine framework the “REPAIRv2” (RNA Editing for Programmable A to I Replacement v2) tool, developed in the laboratory of Dr. Feng Zhang (USA), seems a good alternative to restore the full-length CFTR protein by editing its mRNA containing PTCs. This new approach is based on the possibility of targeting a deaminase enzyme (huADAR2) to a specific Adenosine, to be edited to Inosine (G analogue), on the mutant RNA by a specific guide RNA (gRNA), complementary to the target regions, and a Cas protein. Hypothesis and objectives We applied the new CRISPR/dCas13b based molecular tool of RNA editing (REPAIRv2) to correct the premature stop codon UGA, changing to UGG, in the H2bGFPopal and CFTRW1282X mRNAs with the purpose of recovering the full-length proteins.Essential Methods We designed and cloned the gRNAs needed to target the REPAIRv2 system to the Adenine to be modified. By site-directed mutagenesis we introduced a premature stop codon, W1282X, in the CFTR cDNA. Human HeLa cells expressing the H2BGFPopal mRNA, FRT cells expressing CFTRW1282X and IB3.1 airway epithelial human cells (CFTRΔ508/W12382X) were co-transfected with the plasmids coding for the recombinant protein dCAS13b/ADAR2DD, and for the gRNAs. Fluorescence microscopy was used to analyse the editing results. Results Direct fluorescence microscopy and immunofluorescence analyses detecting the corrected proteins (H2BGFP and CFTR, respectively) suggest that the REPAIRv2 system was able, in different cell lines, to edit the H2BGFPopal and the CFTRW1282X mRNA. However, the rate of editing does not seem high. Indeed, when RNA was purified from transfected cell, retro-transcribed and amplified base correction was not detectable by standard DNA sequencing and western blot. Conclusions Collectively, our results indicate that the REPAIRv2 tool is able to edit the UGA premature stop codon present in the HeLa-H2BGFPopal cells and in engineered FRTW1282X cells harbouring the UGA PTC in the CFTR mRNA. Furthermore, the REPAIRv2 tool worked in the IB3.1 cells suggesting its ability to edit endogenous UGA premature stop codon. Anyway, enhance the delivery of the plasmids as well increase/ stabilize the target mRNA to be edited, seem necessary to improve the efficiency of REPAIRv2. References 1. Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F.- RNA editing with CRISPR-Cas13. Science. 2017 Nov 24; 358 (6366):1019-1027) 2. Lentini L, Melfi R, Di Leonardo A, Spinello A, Barone G, Pace A, Palumbo Piccionello A, Pibiri I. Toward a rationale for the PTC124 (Ataluren) promoted readthrough of premature stop codons: a computational approach and GFP-reporter cell-based assay. Mol Pharm. 2014 Mar 3;11(3):653-64. Acknowledgment FFC#5/2018 funded by FFC and supported by Delegazione FFC di Palermo