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Published on: February 26, 2021
CRISPR/Cas9 system-mediated p21 knockout impairs the MITF signaling pathway
Gyeong Hee Kim1, Moon-Moo Kim2
1Department of Applied Chemistry, Food Science and Technology, Dong-Eui University, Busan 47340, Republic of Korea.
Abstract:
The CRISPR/Cas9 method facilitates targeted disruption of gene sequences, providing a reliable means to analyze gene-dependent regulatory pathways. This study aims to investigate melanogenesis in p21-knockout B16F1 cells generated by the CRISPR/Cas9 system. The mutation was confirmed by DNA Sanger sequencing, which identified frameshift-inducing indels in the p21 locus. The protein structure of p21 in KO cells was predicted by the α-Fold2 and ChimeraX models. The expression level of the p21 gene was completely reduced in RT-PCR and qPCR assays. Notably, while p21-knockout cells exhibited significantly reduced SA-β-galactosidase activity, this was not indicative of cellular rejuvenation. Instead, it correlated with a loss of melanocytic functionality, as evidenced by the concurrent decrease in melanin synthesis and collagen production. Western blotting and immunofluorescence analyses were performed to examine cell cycle and melanogenesis-associated proteins in p21-deficient cells. Loss of p21 resulted in reduced expression of p21, phosphorylated p21, p53, acetylated p53, CDK2, Cyclin D, Cyclin E, MITF, TRP-1, TRP-2, TYR, and p-ERK. Collectively, these findings indicate that p21 is essential for maintaining MITF-driven melanogenic signaling.
Insights
The CRISPR/Cas9 system disrupted the p21 gene, revealing p21 is crucial for melanogenesis, not cellular rejuvenation. Loss of p21 impairs melanin synthesis and melanocytic function.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- The CRISPR/Cas9 system enables precise gene editing for functional genomics.
- Understanding the role of p21 in cellular processes is vital.
Purpose of the Study:
- To investigate the role of p21 in melanogenesis using CRISPR/Cas9-generated knockout cells.
- To analyze the impact of p21 deficiency on melanocytic functionality and associated signaling pathways.
Main Methods:
- CRISPR/Cas9 gene editing to create p21-knockout B16F1 cells.
- DNA Sanger sequencing, RT-PCR, qPCR, Western blotting, and immunofluorescence for molecular analysis.
- Protein structure prediction using α-Fold2 and ChimeraX models.
Main Results:
- CRISPR/Cas9 successfully generated p21-knockout cells with confirmed mutations and complete gene expression reduction.
- p21 deficiency led to decreased SA-β-galactosidase activity, reduced melanin synthesis, and impaired collagen production.
- Loss of p21 downregulated key proteins involved in cell cycle regulation and melanogenesis, including MITF, TRP-1, TRP-2, TYR, and p-ERK.
Conclusions:
- p21 plays an essential role in maintaining MITF-driven melanogenic signaling.
- p21 deficiency negatively impacts melanocytic functionality, contrary to potential implications of reduced SA-β-galactosidase activity.
- This study highlights p21 as a critical regulator of melanogenesis.
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