Genetic modification of cystic fibrosis with ΔF508 mutation of CFTR gene using the CRISPR system in peripheral blood mononuclear cells

Abstract

Objectives: Cystic fibrosis (CF) is an inherited autosomal recessive disease that is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The present study aimed to investigate the genetic modification of CF with ΔF508 mutation of the CFTR gene using CRISPR in peripheral blood mononuclear cells (PBMCs).

Materials and methods: Two single guide RNAs were designed to target sequences in the CFTR gene. The transfection efficiency of PBMC cells was examined through evaluation of green fluorescent protein (GFP) expression using fluorescent microscopy. Moreover, a sgRNA-Cas9 plasmid was tested to target the CFTR gene. The ΔF508 gene modification was evaluated and confirmed by PCR and Sanger sequencing methods.

Results: Our results indicate the feasibility of site-specific gene targeting with the CRISPR/Cas9 system. 33% of the samples were corrected using CRISPR in mutant locus and confirmed by sequence blast at NCBI databases and primers outside the arm locus. CRISPR/Cas9 approach represents an efficient tool to repair the ΔF508 mutation of the CFTR gene in PBMC Cells.

Conclusion: Therefore, the CRISPR system can be highly efficient and specific and provides a powerful approach for genetic engineering of cells and model animals. Generally, the proposed method opens new insights into the treatment of human diseases.

Keywords: CFTR gene; CRISPR; Cystic fibrosis; Peripheral blood - mononuclear cells; ΔF508 mutation.

Figure 1

The location of upstream and downstream primers of the HDR region on chromosome 7 which showed the overlapping regions

Figure 2

Electrophoresis of PCR products on 2% agarose gel in order to find the ΔF508 gene-modified HEK293 cells. (A) The first step product was a 237 bp fragment. (B) The second step product was a 190 bp fragment. No band formation indicates cutting by Cas9

Figure 3

Transfection rate of HEK293 and PBMCs. (A) HEK293 cell transfection with CRISPR vectors. (B) PBMCs before transfection (left), efficiency of PBMCs transfection was about 50%, after 72 hr (middle), efficiency of PBMCs transfection was about 80%, after 120 hr (right)

Figure 4

Transfection rate of PBMC cells using PolyFect after 72 (A) and 120 (B) hr was 50 and 80%, respectively

Figure 5

PBMC isolated from a transfected patient by CRISPR after 120 hr. The modified sequence is shown in the graph

Figure 6

The blast PCR modified product

Figure 7

Specimens amplified a 250 bp product with the HDR downstream primer at temperatures of 55–58 °C. The green sequence represents the HDR sequence, and the red sequence represents the sequence obtained from the amplification of the HDR region downstream, which overlapped the 38 bp with the HDR region. Therefore, the transfected cell modifications were confirmed