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Creation of the Gain-of-Function Mutation of the MITF Gene Related to Melanogenesis Using the CRISPR-Cas9 System

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Abstract

Microphthalmia-associated transcription factor (MITF) is a key transcription factor of Tyrosinase, TRP-1, and TRP-2 genes expression involved in melanogenesis related to aging. The aim of this study was to investigate melanogenesis in MITF gene-mutated human melanocytes edited by CRISPR-Cas9 system. The confirmation of MITF gene mutation was verified using T7E1 assay and Sanger DNA sequencing. Moreover, the gene expression of MITF was investigated using the RT-PCR assay. In addition, Western blot analysis and immunofluorescence staining assay were performed to further validate the difference in protein expression related to melanogenesis between the mutated and normal melanocytes. The MITF gene-edited human melanocytes were created for the first time using the CRISPR-Cas9 ribonucleoprotein complex. The evidence of successful gene editing by the CRISPR-Cas9 ribonucleoprotein complex was confirmed through the identification of mismatch among the MITF gene strands using T7EI assay. Moreover, the mutation region in 75 bp away from DSB sites was analyzed through the Sanger DNA sequencing. The expression levels in both MITF gene and protein were upregulated in mutated melanocytes compared to the normal cells. The expression levels of melanogenesis proteins such as Tyrosinase, TRP-1 and TRP-2 regulated by MITF transcription factor were also remarkably increased in the mutated melanocytes. Above findings provide a clue that the upregulation of melanogenesis could induce in human melanocytes by the edition of the intron in MITF gene using CRISPR-Cas9 ribonucleoprotein complex, leading to the gain of function of MITF gene.

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Funding

This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (no. 2017R1D1A3B06028000).

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Authors and Affiliations

  1. Department of Chemistry and Biology, Dong-Eui University, 614-714, Busan, Korea

    Sojeong Jeon

  2. Department of Applied Chemistry, Dong-Eui University, 614-714, Busan, Korea

    Moon-Moo Kim

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  1. Sojeong Jeon
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  2. Moon-Moo Kim
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Correspondence to Sojeong Jeon or Moon-Moo Kim.

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The authors declare that they have no conflicts of interest. This article does not contain any studies involving animals or human participants performed by any of the authors.

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Jeon, S., Kim, MM. Creation of the Gain-of-Function Mutation of the MITF Gene Related to Melanogenesis Using the CRISPR-Cas9 System. Russ J Genet 58, 857–865 (2022). https://doi.org/10.1134/S1022795422070079

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