FFC#5/2018

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

FFC#5/2018

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

PRINCIPAL INVESTIGATOR

Aldo Di Leonardo (Università degli Studi di Palermo, Dip. di Scienze e Tecnologie Biologiche, Chimiche e Farmaceutiche – STEBICEF)

RESEARCHERS

3

CATEGORY

AREA 1 Therapies to correct the underlying defect

DURATION

1 year

GOAL

€ 21.000 €

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. Collectively, these results indicate that the REPAIRv2 tool is able to edit the UGA premature stop codon present in the HeLa-H2BGFPopal cells and in engineered FRT-W1282X 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.

OTHER RESULTS

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