State Key Laboratory of Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510006, China.
Yi Chuan. 2022 Jul 20;44(7):581-590. doi: 10.16288/j.yczz.22-037.
MC1R (melanocortin 1 receptor) encodes the melanocortin-1 receptor, which can activate intracellular cAMP synthesis under the stimulation of the α-melanocyte stimulating hormone (α-MSH) ligand. Increased cAMP then activates the protein kinase A (PKA) pathway, resulting in the up-regulation of the expression of the microphthalmia-associated transcription factor (MITF) which is a critical regulatory factor of melanin synthesis, and tyrosinase (TYR), the rate-limiting enzyme of melanin synthesis tyrosinase (TYR), and ultimately affects production of eumelanin and pheomelanin, and the coat color phenotype of mammalian species. Previous reports have indicated that the mutation A243T in the transmembrane domain 6 (TM6) of MC1R protein might disrupt the function of MC1R, contributing to the red phenotype in Duroc pig. However, functional analysis of the A243T mutation in MC1R has not yet been carried out. In this study, we attempted to used single-stranded oligo-deoxyribonucleotides (ssODN) as donor templates to introduce the c.727G>A (A243T) mutation into MC1R in human melanoma cell line SK-MEL-2 by CRISPR/Cas9 to analyze its effects on MC1R functions. We found the occurrence of ssODN recombination reached to 10%. Unfortunately, Sanger sequencing MC1R in six single-cell clones revealed that none carried the c.727G>A mutation, but all carried undesired mutations surrounding the target site. Cells transfected with CRISPR/Cas9 plasmids and ssODN presented significantly attenuated cAMP activation, and down-regulated MITF and TYR expression, indicating that the editing MC1R could affect the melanin synthesis function in cells. This study provides a basis for further investigation the mechanism of MC1R mutation on animal coat color.
黑素皮质素 1 受体（MC1R）编码黑素皮质素-1 受体，在α-促黑素细胞激素（α-MSH）配体的刺激下，该受体可以激活细胞内 cAMP 的合成。增加的 cAMP 随后激活蛋白激酶 A（PKA）途径，导致小眼相关转录因子（MITF）的表达上调，MITF 是黑色素合成的关键调节因子，以及酪氨酸酶（TYR），黑色素合成限速酶酪氨酸酶（TYR），最终影响真黑色素和褐黑色素的产生，以及哺乳动物物种的毛色表型。先前的报告表明，MC1R 蛋白跨膜域 6（TM6）中的 A243T 突变可能破坏 MC1R 的功能，导致杜洛克猪的红色表型。然而，MC1R 中的 A243T 突变的功能分析尚未进行。在这项研究中，我们试图使用单链寡脱氧核苷酸（ssODN）作为供体模板，通过 CRISPR/Cas9 将 c.727G>A（A243T）突变引入人黑色素瘤细胞系 SK-MEL-2 中的 MC1R，以分析其对 MC1R 功能的影响。我们发现 ssODN 重组的发生率达到了 10%。不幸的是，对六个单细胞克隆中的 MC1R 进行 Sanger 测序显示，没有一个携带 c.727G>A 突变，但都携带了靶位点周围的非预期突变。转染了 CRISPR/Cas9 质粒和 ssODN 的细胞表现出明显减弱的 cAMP 激活，以及下调的 MITF 和 TYR 表达，表明编辑后的 MC1R 可能会影响细胞中的黑色素合成功能。这项研究为进一步研究 MC1R 突变对动物毛色的机制提供了依据。
