Impact of CRISPR/Cas9 on the expressions of selected immune genes in salmonid cells

Abstract

The discovery of CRISPR/Cas9 as a gene editing tool has revolutionized the field of molecular biology due to its huge potential for innovative applications. However, the biosafety concerns on CRISPR/Cas9-mediated gene editing like off-target mutations, triggering of cell death, and induction of cellular stress need to be elucidated. This thesis aims to investigate the impacts of electroporation and the individual components of CRISPR/Cas9, i.e. sgRNA, Cas9 and RNP complex on ASK-1 cells, using the gene expression of hsp90, hsp70, mhc I, igt and igm as indicators of the impacts. Electroporation was used as a transfection method to edit the cr2 gene and deliver the individual components of CRISPR/Cas9 into the ASK-1 cells. The relative gene expression of the target genes was measured using RT-qPCR and ef-1α was used as the reference gene. The results of this study showed that electroporation as a cell transfection method do not affect the expression of hsp90 and mhc I in ASK-1 cells. On the other hand, the hsp70 was significantly upregulated in Day 2 and Day 7 samples of sham treatment (shocked cells only), however, this might not be due to electroporation since the effect was not seen in other treatments where electroporation was also employed as a mode of transfection. The CRISPR/Cas9 components, sgRNA, Cas9, and RNP complex did not show any effect on the expression of hsp70 and mhc I. Moreover, the expression of hsp90 was not affected by sgRNA and Cas9 components, however it was significantly upregulated in the RNP complex treatment group, both in Day 2 and Day 7 samples. This effect might be the consequence of cr2 gene mutation, as many studies have shown that hsp90 was upregulated during mutations to buffer its lethal effect. The gene expression of igm and igt genes were too low to detect, thus, their gene expression cannot be calculated. To conclude, this study has shown that the electroporation and the CRISPR/Cas9 components did not cause cellular stress nor affected the expression of the immune genes. The impacts that were seen seem to be not attributed to the CRISPR/Cas9-mediated gene editing procedure and components. However, these findings were limited only for the protocols and conditions used in this study using ASK-1 cell line as a model.