Abstract
Gene knockdown and knockout using RNAi and CRISPR/Cas9 allow for efficient evaluation of gene function, but it is unclear how the choice of technology can influence the results. To compare the phenotypes obtained using siRNA and CRISPR/Cas9 technologies, aldehyde dehydrogenase 2 (ALDH2) was selected as an example. In this study, we constructed one HepG2 cell line with a homozygous mutation in the fifth exon of ALDH2 (ALDH2-KO1 cell) using the eukaryotic CRISPR/Cas9 expression system followed by the limited dilution method and one HepG2 cell line with different mutations in the ALDH2 gene (ALDH2-KO2 cell) using the lentivirus CRISPR/Cas9 system. Additionally, one ALDH2-knockdown (KD) HepG2 cell line was created using siRNA. The reproducibility of these methods was further verified in the HEK293FT cell line. We found that the mRNA expression level of ALDH2 was significantly decreased and the protein expression level of ALDH2 was completely abolished in the ALDH2-KO cell lines, but not in ALDH2-KD cells. Furthermore, the functional activity of ALDH2 was also markedly disrupted in the two ALDH2-KO cell lines compared with ALDH2-KD and wild-type cells. The lack of ALDH2 expression mediated by CRIPSR/Cas9 resulted in a more dramatic increase in the cellular susceptibility to chemical-induced reactive oxygen species generation, cytotoxicity, apoptosis, and inflammation, especially at low concentrations compared with ALDH2-KD and WT cells. Therefore, we consider the gene knockout cell line created by CRISPR/Cas9 to be a more useful tool for identifying the function of a gene.
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We thank Nature Research Editing Service for its linguistic assistance during the preparation of this manuscript.
Funding
This study was funded by the National Natural Science Foundations of China (Grant numbers: 81273098, 81673140, 81402715), Science and Technology Planning Project of Guangzhou City (Grant number: 201707010078), Young teacher training Program of Sun Yat-sen University (Grant numbers: 15ykpy06, 14ykpy06), A Major Scientific Project of NSFC (Grant number: 91543208).
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Wang, F., Guo, T., Jiang, H. et al. A comparison of CRISPR/Cas9 and siRNA-mediated ALDH2 gene silencing in human cell lines. Mol Genet Genomics 293, 769–783 (2018). https://doi.org/10.1007/s00438-018-1420-y
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DOI: https://doi.org/10.1007/s00438-018-1420-y