Microscope by Michael Longmire
Proteins provide a wide array of functions for cells, as we’ve discussed in previous articles, and as such pose a challenge for accurately describing their functions and interactions on the whole. This accounts for issues like the protein folding problem and the lack of other predictions of proteins. As it turns out, our popular gene editing tool CRISPR is able to help uncover the machinations of proteins. Using a CRISPR Cas complex system, which we explained in this article, the coding gene for a protein can be intentionally mutated to effect changes in the structure. By observing these changes scientists can learn about what part of the amino acid structure conveys a function to the overall protein. These functions can vary between enzymatic and non-enzymatic which can be summarized by reaction catalyzing and interaction dynamic processes respectively.
Vinyard et. Al. set out to test CRISPR’s ability to create those changes and to observe cell viability caused by the changes in the gene. When the genes were altered too much or in the wrong areas, the cells would die off leaving only viable mutations of genes. It is imperative that they first determine if CRISPR is in fact causing the changes they are dependent on so that they can make accurate conclusions about the protein in question. To do this, they target lysine-specific histone demethylase 1 (LSD1), a protein involved in the regulation of DNA expression, in the context of acute myeloid leukemia (AML) cell lines SET-2 and MV4;11. Typically LSD1 helps regulate transcription of genes by demethylating histones, the proteins DNA wrap around, which generally serves to decrease expression of the affected DNA. In cases of AML, scientists had believed that it was this process that was allowing the immature white blood cells to continue dividing. Upon further inspection however, Vinyard and colleagues hypothesize that there are other non-enzymatic processes at play that confer the reproduction and growth qualities of AML. In this case, the enzymatic process of LSD1 is the removal of methyl groups from histones while other non-enzymatic processes would include co-factor binding, regulative binding, and many others.
With this protein test subject they use targeted PAM-restricted and control single guide RNA (sgRNA) to direct the CRISPR-Cas system to specific sites along the LSD1 gene. Essentially they mutate the sites on the gene they suspect are important for the structure and therefore function of the protein as well as other regions that present similar target viability to find new protein interactions. They compare the two types of sgRNA data to see if they produce significant observable changes in the protein structure that can elucidate the additional functions of LSD1. To observe concrete changes to the structure they use GSK-LSD1, a known inhibitor of LSD1 which seems to help treat AML by decreasing growth, to find the protein region which binds to the inhibitor. They find that GSK-LSD1 binds to LSD1 in the enxymatic region of the protein, as suspected, stopping LSD1 from demethylating DNA histones. Additionally, they are able to show that varying versions of this inhibitor GSK-LSD1 produce distinguishable changes between each other. All of this helps show that CRISPR is able to not only find functional regions on the protein confirmation but to distinguish between structural-activity relationships (SARs).
Given that this process suggested to the team that there are a lot of enzymatic changes possible, they wanted to see if all of those changes are even relevant to the growth of AML cell lines. To do this they develop numerous SET-2 AML mutants with GSK-LSD1 treatments to see which groups of cells could continue multiplying the immature leukocytes. These mutants generally had no enzymatic activity meaning the DNA was different. Despite this the cells were still dividing as normal which suggested to the team that there were other factors at play. Their data thus far as well as other research articles suggested that protein binding interactions must be what conveys the real growth signals. In particular, other papers hypothesized the inhibitors involvement in preventing LSD1 from interacting with GFI1B. They are able to use CRISPR scanning to show that it is in fact not only this protein interaction that conveys some proliferative qualities to AML leukocytes, but the inhibitors themselves tend to induce expression of other factors that change DNA expression along with decreased LSD1 activity and protein binding.
The paper shows that CRISPR is useful in yet another area of study and proves to be a multifunctional tool for research. CRISPR scanning can help understand new functions of discovered and future proteins that aren’t readily apparent. By being able to tweak the structure relatively precisely one can expect more protein gene therapies and clinical treatments for disorders. The first step in being able to treat any disease or disorder is understanding of the faulty mechanism. While a lot of optimization and improvements on efficiency can be made, CRISPR is able to deconvolute protein interactions along with providing gene editing capabilities. On the whole it seems that GSK-LSD1 inhibits LSD1 through a non-enzymatic interaction with GFI proteins which complex to affect transcriptions and regulations in the cell. This information presents a new understanding of LSD1 and shows that CRISPR can successfully be modified to target very particular genes for research purposes outside of direct treatments and such.
In Depth
The research presented in this article is extensive and exact. A lot of work was done to collect the relevant data and it shows, however this does not come with added explanation. The data presented covers a wide range of research areas and it is to be expected that many more questions arise from a study like this. The team do make a point to include potential future directions for the research which is highly beneficial and inspiring. In particular they mention increasing the resolution of CRISPR mutagenesis and applications to clinical settings as areas for further research. I find it very interesting that the research conducted is a new age positive selection similar to replica plating which makes use of viable proteins rather than treatments to determine survivors. The methods for this research was laid out more directly than the rest of this paper and that is appreciated. This research is some of the first unanticipated applications of CRISPR Cas systems I have experienced and am excited to see where this can be taken. I am wary of the continued complicated jargon and lack of explanation as it would contribute to the limitation of new minds contributing to the research or even thoughts on the matter. Overall, this paper makes good conclusions with solid evidence but lacks sorely in explanations and directing the readers attention to the reasoning and logical determinations of why this protein LSD1 is suspected to interact with GFI1B for example and how they influence expressions in the cell.
Vinyard, M. E., Su, C., Siegenfeld, A. P., Waterbury, A. L., Freedy, A. M., Gosavi, P. M., … & Bauer, D. E. (2019). CRISPR-suppressor scanning reveals a nonenzymatic role of LSD1 in AML. Nature Chemical Biology, 15(5), 529.
Michael Longmire.https://unsplash.com/@f7photo
National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/
Unsplash. https://unsplash.com
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