CRISPR/Cas9 mediated gene knockout of Glb1 gene in mouse embryonic fibroblasts

Abstract

BackgroundThe occurrence of mutations at different locations in the Glb1 gene in nerve cells can lead to the deficient activity of a hydrolase enzyme called β-galactosidase 1 in the lysosomal organelle. Therefore, the substrates of this enzyme (gangliosides) aggregate in the cells at toxic levels, leading to the progressive degeneration of nerve cells. Since humans are incapable of repairing the damaged nerve cells, this in itself causes a rare genetic disorder called GM1 gangliosidosis. Doctors are only able to reduce the signs of this disease, and technically, no effective treatment exists capable of eliminating its cause. Recently, using methods based on genome editing, aiming to achieve a more accurate understanding of the basic cellular and molecular mechanisms leading to this disease and find new therapeutic solutions. MethodsIn the current study, specific guide RNAs was designed for exons 2 and 6 of the Glb1 gene, and entered these gRNAs into lenticrispr expression vector containing CRISPR elements. Then, mouse embryonic fibroblast (MEF) cells transferred with recombinant vectors containing our gRNAs. Finally, the generated mutations were investigated via the polymerase chain reaction (PCR) test. ResultsOur results confirmed correct transformation process through colony PCR and Sanger sequencing. Then, by performing PCR test on genomic DNA of puromycin resistant MEF cells, a product band of 824 bp was observed that indicated successful knockout of the Glb1 gene. ConclusionsThe current study was designed as a proof of concept to show possibility of knocking out the Glb1 gene, using designed sgRNAs.