Since its introduction more than ten years ago, CRISPR technology has been used in a remarkable range of applications from biomedicine to food production. Now, a team of scientists from Stanford University have used it to uncover a list of genes involved in producing and regulating human skin pigmentation. Their research highlighted 135 previously unknown so-called melanin-promoting genes and characterized the roles of two proteins involved in making melanocytes—the specialized cells that produce melanin. Insights from the study could help improve scientists’ understanding of skin cancer, skin pigmentation defects, and even neurodegenerative disease.

Details of the study were published in Science in a paper titled “A genome-wide genetic screen uncovers determinants of human pigmentation.”

The rich diversity of human skin tones is determined by the quantity, type, and distribution of melanin pigment present in the individual. In the past, scientists have done genome-wide association studies focused on melanocytes that have, among other things, mapped genes involved in hypo- and hyperpigmentation diseases. But the exact nature of the genetics of skin pigmentation has not been fully explored, according to the researchers. In fact, results from prior GWAS studies only account for a small fraction of the observed variation in the populations used for those studies.

Understanding the genetics of variation in human traits has been a long-standing goal of the lab of Joanna Wysocka, PhD, a professor of developmental biology and chemical and systems biology at Stanford University and the senior author on the paper. Her lab’s work includes studies of craniofacial differences in people and cell types which give rise to them. These cells emerge from neural crest cells during early development as do melanocytes, which are responsible for variation in skin pigmentation.

This study marks the first time CRISPR has been used to explore the genes that work in regulating human melanin production and skin pigmentation, according to Wysocka. “For the first time, we could do a whole-genome genetic screen in human cells,” she said. “[With CRISPR] what became possible, we could just dream about before.” Without it, the screening step of this study would have been much more challenging to do.

Vivek Bajpai, PhD, now an assistant professor of chemical, biological, and materials engineering at University of Oklahoma and lead author on the paper, worked on the current study while he was completing a post-doctoral program in Wysocka’s lab at Stanford. Some of his research interests are in stem cell and tissue engineering including the use of induced pluripotent stem cells to create neural crest cells and melanocytes. His knowledge of the ways that these cells interact with light was instrumental in designing the assay the team used to distinguish between differently pigmented cells. “The assay had to be relatively simple so that we could scale it up. This is where Vivek’s idea with using the light scatter properties came in,” Wysocka said.

Pigmented melanocytes scatter light in different ways depending on the level of melanin present. And those scatter profiles can be detected using flow cytometry. Relying on his knowledge of this feature, Bajpai developed a flow cytometry-based assay capable of separating darker melanocytes from lighter ones based on their scatter profiles. This crucial first step made it possible for them to perform genome-wide CRISPR screens on groups of similar cells that ultimately allowed them to assess the impact of different genes on melanin.

As Bajpai explained, the team designed the screen to identify only genes directly involved in making melanin and ignore those that suppress it. And there were other constraints. Since they were using differentiated melanocytes, the screen left out any genes that affect pigmentation at the stages of development prior to maturity. The screen also excluded any genes that are essential to cell survival and play a role in pigmentation, the researchers wrote.

The screen identified 169 genes involved in melanogenesis including several known ones. Excluding the known genes left 135 that were not previously associated with skin color in other studies. “We hypothesized that if these genes were involved in making more melanin, [when] we compare dark vs light melanocytes, the genes will [likely]be expressed at a higher level,” Bajpai said. But there is an important caveat that is explained in the paper.

Variations in melanin levels could be due to genetic encoding or the result of different rates of melanogenesis resulting in differential expression of melanogenesis genes. More pigmented melanocytes typically have higher rates of melanogenesis, which may require higher levels of expression of genes that are directly or indirectly involved in pigment production. To try to assess the impact of genetic encoding involving the genes identified by their screen, the scientists tested RNA levels in foreskin samples from 33 newborns with varying melanin levels. The results showed that most genes that their screen identified were upregulated in newborns that had darker skin tones.

But these differences in RNA levels between darkly and lightly pigmented melanocytes could be due to genetic encoding or a consequence of different melanogenesis rates. So, the team dug deeper and compared their findings to data from other genome-wide association studies. They found that some of the genes their screen identified were associated with variation in skin tones in those populations suggesting that genetic encoding plays a significant role in the observed melanin levels.

There is much to learn about exactly how the genes identified by the screen contribute to melanogenesis but there are some broad themes. The scientists categorized the 135 candidate genes into two groups: those involved with gene regulation and those involved with endosomal trafficking pathways. As part of the study, they picked one example from each group and characterized their underlying mechanisms in more detail. Those results are also reported in the paper.

Specifically, they selected a transcription factor called KLF6, and confirmed its involvement in regulating the expression of some other melanin-associated genes, and in skin pigmentation in mouse models. From the endosomal trafficking cohort, they focused on a protein coding gene called COMMD3. This gene plays a part in regulating the pH of melanosomes which is important for the activity of tyrosinase, a major enzyme in melanin synthesis. Tyrosinase is responsible for the first step in melanin production—converting tyrosine into dopaquinone which is then converted into melanin through various chemical reactions. Deleting COMMD3 lowers the pH of the melanosomes and tyrosinase becomes inactive resulting in defective pigmentation.

Findings like these provide ample opportunities for other research groups to build on, including conducting basic research into human variation as well as disease-focused studies.

Now running his own lab in Oklahoma, Bajpai hopes to apply these findings to diseases like cancer. And he believes an interesting angle will be to study correlations between skin pigmentation levels and melanoma susceptibility. “There is a UV angle to melanoma where if you have less melanin, your skin melanocytes are less protected and likely to mutate faster—that’s established,” he said. “[But] it turns out that there is an inverse relationship between the level of melanin in the skin and melanoma initiation in those cells. Melanin levels affect how fast cells can divide and so we are trying to follow up some of the new hits in the context of melanoma initiation.” His team will also explore how some of the genes influence variation in human pigmentation and melanogenesis. And they will apply the approach to non-human organisms as well, he said.

Unsurprisingly, the findings reported here could benefit research into instances of hypo and hyper-pigmentation such as albinism and vitiligo. But another intriguing potential area of interest from Wysocka’s point of view is in the brain.

Specifically, scientists could explore the role of these genes, if any, in the making of neuromelanin, a form of melanin produced by neurons in the substantia nigra region of the brain. The demise of cells in this area are associated with the development of Parkinson’s disease. There is some evidence that neuromelanin has a protective value in the brain, and indeed some research into Parkinson’s patients shows that they have lower levels of neuromelanin. Future studies could explore if the same genes involved in skin pigmentation are at work in the brain and to what extent, Wysocka said.

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