Application of CRISPR/Cas9 system for generation of MCD disease cell model
Iman Safari1 , Elahe Elahi1 *, Shohreh Issazadeh-Navikas2 , Alireza Baradaran-Rafii3
- School of Biology, University College of Science, University of Tehran, Tehran, Iran
- Neuroinflammation Unit, Biotech Research and Innovation Centre (BRIC), University of Copenhagen, Copenhagen, Denmark
- Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran
Abstract: The potential of the CRISPR/Cas9 tool for generation of disease models is one of its many applications. Macular corneal dystrophy (MCD) is a recessive disorder caused by CHST6 mutations. We aimed to generate a cell model for MCD using the CRISPR/Cas9 system to target and disrupt its causative gene.
Methods: The literature was reviewed to identify suitable target regions within CHST6. The criteria used included prevalence of mutations within the regions to be chosen among Iranian patients and patients of other countries. To this end, mutation screening was performed for 21 MCD-affected Iranians. Bioinformatics tools were applied for design of guide RNAs (sgRNAs). The chosen sgRNA molecules were cloned into a px458 construct. The efficiency of the sgRNAs was initially assessed using HEK293FT cells and T7E1 mismatch cleavage assay. Two different lentiviral particles for transfer of the most efficient sgRNA and the spCas9 gene were produced. Corneal keratocyte cells, the most biologically relevant cells to MCD pathogenesis, were transfected using the sgRNA containing px458 vector. HEK and keratocyte cells were also induced separately using virus particles. The efficiency of Cas9 expression was investigated using by Western blotting.
Results: A 36 nucleotide region (c.598-c.633) in CHST6 that encodes amino acids 200-211 was chosen as the target region because of evident clustering of mutations therein. Two sgRNAs were designed for the target region. The T7E1 assay showed that one of the sgRNAs functioned efficiently in HEK cells. Keratocyte cells were resistant to transfection via different protocols. The engineered lentiviruses that contained sgRNA and spCas9 encoding genes worked efficiently in genome modification of the HEK cells, but not in the biologically relevant keratocytes. Cas9 expression was confirmed in both cell types.
Conclusion: Although notable success of CHST6 gene modification using the CRISPR/Cas9 system was achieved in HEK cells, neither plasmid nor lentivirus vectors worked efficiently in keratocyte cells. The efficiency of sgRNA molecules may differ in different cell types. A more serious consideration is the possible existence of specific and strong DNA repair mechanisms in corneal cells. Such systems may have evolved in response to prolonged exposure to ultraviolet mutagenesis.
