Gene-editing taken to advanced levels in human stem cells

Gene-editing taken to advanced levels in human stem cells

Researchers create new brain cancer model using genetically engineered pluripotent stem cells The green glow thus signifies a better than 90% chance that the cell has been successfully edited, allowing the researchers to pick out edited cells more efficiently and leave out the others. The edited (green) cells are then cultured by themselves, to produce clones that can be grown indefinitely to produce a renewable pool of cells with the particular gene editing that was needed. TREE- the advantages The disease process in AD is complex, not due to a single base mutation unlike sickle cell anemia or cystic fibrosis. There could be multiple factors that act in synchrony to increase the risk of the disease. Brafman explains the advantage of using TREE in this situation: “We wanted a way to introduce multiple edits simultaneously in pluripotent stem cells. Because otherwise, you would have to take this sequential iterative approach, where you introduce one edit, isolate a clonal population introduce another edit, and so on.” For their study the team used induced pluripotent stem cells derived from both healthy and AD patients, the latter having either sporadic or late-onset AD. These cells represent the nerve cells and other cell types found in the brain in those patients who have these risk factors, since they have the same DNA. The APOE gene was the target of the current single-base proof-of-concept gene editing experiment, This gene comes in three variants, one called the APOE4 which increases the risk of late-onset AD. Using TREE, the scientists edited single bases in this gene, and unlike CRISPR, they could accurately modify both copies of the gene. Now, says Brafman, “We can understand why an APOE variant can increase or decrease risk, and then we can start targeting those pathways that are affected.” Another advantage of TREE is the ability to knock out, or remove, genes of importance to the disease process. This will allow scientists to see if the gene (in this case, APOE4) is good for the cell, bad for it, or doesn’t make a major difference at all. “The traditional CRISPR approach is that you have to edit once to get a heterozygous edit, then isolate that clone, edit again to get another heterozygous edit," according to Brafman. "So, it's very inefficient in that way. We are generating homozygous edits at an efficiency approaching 90%. I haven't seen any other technologies that can do that in pluripotent stem cells.” The findings The results are astounding: the researchers, even though not from a laboratory that specializes in gene editing, have managed to edit single bases in the DNA strand with extreme accuracy and up to 90% efficiency, using human stem cells. Formerly, the scientists had no idea whether the desired edits had been made because the cells did not show any difference. Instead of making what Brafman terms “a random guess”, poking CRISPR at cells and hoping to get the right results, with about 10% to 15% accuracy, they are able to pick out edited cells and basically go ahead with just those cells. Previous research by this team showed the potential of the TREE approach in human cell gene editing. The current thrust was on further refining it to achieve quick and efficient human stem cell editing as well. They succeeded in using TREE to generate stem cell populations in which multiple genes had been edited simultaneously. Over 80% of the cells showed successful edits at all three target locations, and in all these cases the edits had been made on both strands. In other words, a multiplex approach allowed them to reach the same high efficiency as making sequential edits one by one, while obtaining a pool of in vitro cells in which the disease process can be studied, and drugs can be tested out. Conclusion Brafman says, “We want to keep expanding on that toolbox. We've already gotten a high level of interest from other scientists who will be using this to generate their own cell lines. We envision this method will have important implications for the use of human stem cell lines in developmental biology, disease modeling, drug screening and tissue engineering applications.” Journal reference: BIG-TREE: Base-Edited Isogenic hPSC Line Generation Using a Transient Reporter for Editing Enrichment Brookhouser, Nicholas et al. Stem Cell Reports, https://www.cell.com/stem-cell-reports/fulltext/S2213-6711(19)30452-7



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