Investigating REPAIRv2 as a Tool to Edit CFTR mRNA with Premature Stop Codons

Abstract

Cystic fibrosis (CF) is caused by mutations in the gene encoding the transmembrane conductance regulator (CFTR) protein. Some CF patients are compound heterozygous or homozygous for nonsense mutations in the CFTR gene. This implies the presence in the transcript of premature termination codons (PTCs) responsible for a truncated CFTR protein and a more severe form of the disease. Aminoglycoside and PTC124 derivatives have been used for the read-through of PTCs to restore the full-length CFTR protein. However, in a precision medicine framework, the CRISPR/dCas13b-based molecular tool "REPAIRv2" (RNA Editing for Programmable A to I Replacement, version 2) could be a good alternative to restore the full-length CFTR protein. This RNA editing approach is based on the targeting of the deaminase domain of the hADAR2 enzyme fused to the dCas13b protein to a specific adenosine to be edited to inosine in the mutant mRNA. Targeting specificity is allowed by a guide RNA (gRNA) complementarily to the target region and recognized by the dCas13b protein. Here, we used the REPAIRv2 platform to edit the UGA PTC to UGG in different cell types, namely IB3-1 cells, HeLa, and FRT cells engineered to express H2BGFP^opal and CFTR^W1282X, respectively.

Keywords: CRISPR/dCas13b; RNA editing; cystic fibrosis; premature termination codons (PTCs).

Figure 1

Schematic representation of mRNA editing by REPAIRv2; sgRNA: Specific guide RNA. The C:A mismatch at the target position and the outcome of editing, A to I replacement in the mRNA, are shown.

Figure 2

Fluorescence analysis to detect editing of the PTC (UGA) in HeLa cells expressing the H2BGFP^opal gene. HeLa-H2BGFP^opal cells were co-transfected with dCAS13b/ADAR2_DD and selected gRNAs coding plasmids (450 ng total DNA). Two days after transfection, cells were fixed with methanol for 5 min and the green fluorescence due to H2BGFP expression was monitored using the 63X objective on a ZEISS microscope equipped for epifluorescence. Nuclei were visualized with DAPI (blue). HeLa H2BGFP^WT cells and untransfected H2BGFP^opal cells were used as positive and negative control, respectively.

Figure 3

Fluorescence analysis after editing the PTC (UGA) in HeLa-H2BGFP^opal cells stably expressing dCAS13b/ADAR2_DD. HeLa cells stably expressing dCAS13b/ADAR2_DD were transfected with selected gRNAs coding plasmids (300 ng of total DNA). Two days after transfection, cells were fixed with methanol for 5 min and the H2BGFP (green) was detected using the 63x objective on a ZEISS microscope equipped for epifluorescence. Nuclei were stained with DAPI (blue). HeLa-H2BGFP^WT cells and untransfected HeLa-H2BGFP^opal cells, expressing dCAS13b/ADAR2_DD, were used as positive and negative control, respectively.

Figure 4

H2BGFP gene expression levels measured by RT-qPCR in HeLa-H2BGFP^WT and H2BGFP^opal cells. The histogram represents the mean ± SD of a quadruplicate.

Figure 5

CFTR gene expression level evaluated by RT-qPCR in FRT-CFTR^WT and FRT-CFTR^W1282X stably transfected cells. The histogram represents the mean ± SD of a quadruplicate.

Figure 6

Immunofluorescence detection of CFTR protein in FRT-CFTR^W1282X cells. Cells untransfected (negative control) and transfected with the plasmids encoding the indicated gRNA and Cas13b/ADAR2_DD (300 ng total DNA) are shown. FRT-CFTR^WT cells were used as a positive control. Cells were fixed with methanol and the CFTR protein was revealed by the primary antibody mAbCF3, followed by a secondary antibody anti-mouse Alexa-488, (green, Abcam). Nuclei (blue) were DAPI stained. Images were taken at 100x magnification on a ZEISS microscope equipped for epifluorescence.

Figure 7

Immunofluorescence analysis of CFTR protein in triton permeabilized FRT-CFTR^W1282X cells and quantification of the immunofluorescence. FRT-CFTR^W1282X cells were transfected with the plasmids encoding the indicated gRNAs and dCAS13b/ADAR2_DD (300 ng total DNA). Untransfected FRT-CFTR^W1282X cells (NT) were used as negative control. FRT-CFTR^W1282X cells treated with G418 and FRT-CFTR^WT cells were used as a positive control. (A) The CFTR protein was revealed by the primary antibody mAb570, followed by a secondary antibody anti-mouse-FITC conjugated (green, Sigma). Nuclei (blue) were DAPI stained. Images were taken at 63x magnification on a ZEISS microscope equipped for epifluorescence. (B) The quantification is relative to the replicate representative of the results. The error bar represents the standard error of the mean (SEM). The quantification of CFTR signal was done manually by using Fiji software. The cell contour was drawn by using a cell membrane marker. The background was subtracted and the integrated signal intensity in the selected area (one cell) was measured.

Figure 7

Immunofluorescence analysis of CFTR protein in triton permeabilized FRT-CFTR^W1282X cells and quantification of the immunofluorescence. FRT-CFTR^W1282X cells were transfected with the plasmids encoding the indicated gRNAs and dCAS13b/ADAR2_DD (300 ng total DNA). Untransfected FRT-CFTR^W1282X cells (NT) were used as negative control. FRT-CFTR^W1282X cells treated with G418 and FRT-CFTR^WT cells were used as a positive control. (A) The CFTR protein was revealed by the primary antibody mAb570, followed by a secondary antibody anti-mouse-FITC conjugated (green, Sigma). Nuclei (blue) were DAPI stained. Images were taken at 63x magnification on a ZEISS microscope equipped for epifluorescence. (B) The quantification is relative to the replicate representative of the results. The error bar represents the standard error of the mean (SEM). The quantification of CFTR signal was done manually by using Fiji software. The cell contour was drawn by using a cell membrane marker. The background was subtracted and the integrated signal intensity in the selected area (one cell) was measured.

Figure 8

Immunofluorescence analysis to detect the CFTR protein in triton permeabilized IB3-1 cells and quantification of the immunofluorescence. IB3-1 cells were untreated (NT: Negative control), treated with G418 (positive control), or transfected with the plasmids encoding the 50-32/5034 gRNAs and dCAS13b/ADAR2_DD. (A) CFTR protein was revealed by the specific primary antibody mAb570 and a secondary antibody (green, Alexa-488, Abcam). Nuclei (blue) were DAPI stained. Images were taken at 63x magnification on a ZEISS microscope equipped for epifluorescence. (B) The quantification is relative to the replicate representative of the results. The error bar represents the SEM. The quantification of CFTR signal was done manually by using Fiji software. The cell contour was drawn by using a cell membrane marker. The background was subtracted and the pixel mean in the selected area (one cell) was measured.

Figure 9

Electropherograms of sequenced H2BGFP^opal and CFTR^W1282X amplicons from cells expressing (A) GFP gRNA 50–34, (B) GFP gRNA 50–35, (C) CFTR gRNA 50–34. Mutant positions are indicated by the arrows.

Figure 10

Electropherograms relative to H2BGFP WT/opal amplicons’ mix at 1:1 (A) and 2:1 ratio (B). The double peaks are indicated by the arrows.