Class
Article
College
College of Science
Department
English Department
Faculty Mentor
Irina Polejaeva
Presentation Type
Poster Presentation
Abstract
Cystic Fibrosis (CF) is a human genetic disease caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. Among the ~2000 known CF mutations, F508del mutation is found in 84% and G542X in 4.6% of the CF patients in US, respectively. The F508del mutation occurs in the exon 11 and is characterized by the deletion of the ‘CTT’ nucleotides that ultimately deletes the Phenylalanine residue at the position 508 of CFTR. It causes the misfolding of the CFTR protein, which is further degraded by proteases. The G542X is a nonsense mutation found in the exon 12 and associated with nonsense-mediated decay of the mutant transcript causing the absence of protein production. Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and SCNT techniques. We previously hypothesized that gene editing may be an effective tool to correct these mutations and permanently cure this genetic disease. Thus, in a previous study, we evaluated the efficiency of CRISPR/Cas9-meditated gene knock-in to correct the F508del and G542X mutations in sheep fibroblasts in vitro. In total, we isolated 32 single cell bacterial colonies for each of the mutant. Sequencing results indicate that ‘CTT’ was introduced in 4/26 (15.3%) plasmid colonies, and ‘T to G’ replaced in 13/31 (41.9%) colonies. Therefore, our results indicate that the F508del and G542X mutations can be effectively corrected in CF sheep fibroblasts in vitro using CRISPR/Cas9 approach. Due to the high percentage of indels in the correction of G542X (13%) and F508del (64%) we have hypothesized that the use of a piggyBac transposon may reduce the rate of indels and increase the efficiency of correction of mutations. While ongoing we have continued to successfully integrate the various components necessary to construct the transposon prior to testing the impacts on bacterial colonies.
Location
Logan, UT
Start Date
4-8-2022 12:00 AM
Included in
Correction of F508del and G542X Mutations in Sheep Fibroblasts of Cystic Fibrosis Models Using CRISPR/Cas9 and PiggyBac
Logan, UT
Cystic Fibrosis (CF) is a human genetic disease caused by mutations in the CF Transmembrane Conductance Regulator (CFTR) gene. Among the ~2000 known CF mutations, F508del mutation is found in 84% and G542X in 4.6% of the CF patients in US, respectively. The F508del mutation occurs in the exon 11 and is characterized by the deletion of the ‘CTT’ nucleotides that ultimately deletes the Phenylalanine residue at the position 508 of CFTR. It causes the misfolding of the CFTR protein, which is further degraded by proteases. The G542X is a nonsense mutation found in the exon 12 and associated with nonsense-mediated decay of the mutant transcript causing the absence of protein production. Previously, we generated CFTRF508del/F508del and CFTRG542X/G542X lambs (unpublished) using CRISPR/Cas9 and SCNT techniques. We previously hypothesized that gene editing may be an effective tool to correct these mutations and permanently cure this genetic disease. Thus, in a previous study, we evaluated the efficiency of CRISPR/Cas9-meditated gene knock-in to correct the F508del and G542X mutations in sheep fibroblasts in vitro. In total, we isolated 32 single cell bacterial colonies for each of the mutant. Sequencing results indicate that ‘CTT’ was introduced in 4/26 (15.3%) plasmid colonies, and ‘T to G’ replaced in 13/31 (41.9%) colonies. Therefore, our results indicate that the F508del and G542X mutations can be effectively corrected in CF sheep fibroblasts in vitro using CRISPR/Cas9 approach. Due to the high percentage of indels in the correction of G542X (13%) and F508del (64%) we have hypothesized that the use of a piggyBac transposon may reduce the rate of indels and increase the efficiency of correction of mutations. While ongoing we have continued to successfully integrate the various components necessary to construct the transposon prior to testing the impacts on bacterial colonies.