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

Risultato della ricerca: Conference articlepeer review


Background and RationaleSome CF patients are compound heterozygous or homozygous fornonsense mutations in the CFTR gene. Mutant CFTR gene coding fortranscripts with premature termination codons (PTCs) is responsiblefor truncated CFTR protein and for a severe form of the disease. In aprecision medicine framework the “REPAIRv2” (RNA Editing forProgrammable A to I Replacement v2) tool, developed in the laboratoryof Dr. Feng Zhang (USA), seems a good alternative to restorethe full-length CFTR protein by editing its mRNA containing PTCs.This new approach is based on the possibility of targeting a deaminaseenzyme (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 objectivesWe applied the new CRISPR/dCas13b based molecular tool of RNAediting (REPAIRv2) to correct the premature stop codon UGA, changingto UGG, in the H2bGFPopal and CFTRW1282X mRNAs with thepurpose of recovering the full-length proteins.Essential MethodsWe designed and cloned the gRNAs needed to target the REPAIRv2system to the Adenine to be modified. By site-directed mutagenesiswe introduced a premature stop codon, W1282X, in the CFTR cDNA.Human HeLa cells expressing the H2BGFPopal mRNA, FRT cells expressingCFTRW1282X and IB3.1 airway epithelial human cells(CFTRΔ508/W12382X) were co-transfected with the plasmids codingfor the recombinant protein dCAS13b/ADAR2DD, and for the gRNAs.Fluorescence microscopy was used to analyse the editing results.ResultsDirect fluorescence microscopy and immunofluorescence analysesdetecting the corrected proteins (H2BGFP and CFTR, respectively)suggest that the REPAIRv2 system was able, in different cell lines, toedit the H2BGFPopal and the CFTRW1282X mRNA. However, the rateof editing does not seem high. Indeed, when RNA was purified fromtransfected cell, retro-transcribed and amplified base correction wasnot detectable by standard DNA sequencing and western blot.ConclusionsCollectively, our results indicate that the REPAIRv2 tool is able to editthe UGA premature stop codon present in the HeLa-H2BGFPopalcells and in engineered FRTW1282X cells harbouring the UGA PTC inthe CFTR mRNA. Furthermore, the REPAIRv2 tool worked in the IB3.1cells suggesting its ability to edit endogenous UGA premature stopcodon. Anyway, enhance the delivery of the plasmids as well increase/stabilize the target mRNA to be edited, seem necessary to improvethe efficiency of REPAIRv2.References1. 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, PalumboPiccionello A, Pibiri I. Toward a rationale for the PTC124 (Ataluren)promoted readthrough of premature stop codons: a computationalapproach and GFP-reporter cell-based assay. Mol Pharm. 2014 Mar3;11(3):653-64.Acknowledgment FFC#5/2018 funded by FFC and supported by DelegazioneFFC di Palermo
Lingua originaleEnglish
pagine (da-a)7-7
Numero di pagine1
RivistaBMC Pulmonary Medicine
Stato di pubblicazionePublished - 2020


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