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