Gene-editing taken to advanced levels in human stem cells

By Dr. Liji Thomas, MDJan 30 2020

A new study published in the journal Stem Cell Reports in January 2020 reports the use of an adaptation to a commonly used gene editing technology to achieve a more than 900% increase in efficiency.

CRISPR spells progress in gene therapy

Over the past 10 years or so, the revolutionary gene editor CRISPR has changed most of the rules in the area of genetic research. Scientists have been exploring the reach of this incredibly useful tool since then. In fall 2019, the first clinical trials began with the use of CRISPR to change around genes that cause cancer, for instance. The fundamental principle of this therapy is to extract some cells from the subject, edit the target gene using CRISPR, and re-insert the edited cells into the same individual to allow them to combat the disease. This looks like the ultimate dream of perfectly personalized medicine which can actually correct disease right from the root.

3D illustration of CRISPR-Cas9 genome editing system. Image Credit: Meletios Verras / Shutterstock

The problems with CRISPR

However, CRISPR is still in a comparatively nascent state. Safety issues exist with CRISPR use, including the danger that it may edit unwanted bits of DNA or target the wrong sites, leading to the so-called “off-target” gene effects.

Another problem is that scientists haven’t really mastered the art of getting CRISPR editing to do what it ought to do in all cells. So far, only about 10% of experiments succeed in achieving the desired editing, whichever the cell targeted.

Most CRISPR editing has been carried out on immortalized cell lines or cancer cells lines, in which editing is easier to do. However, pluripotent stem cells are much more useful in genetic research but are harder to modify. As a result, scientists are still pursuing more successful and efficient gene editing, especially in stem cells.

About stem cells

Induced pluripotent stem cells are healthy adult cells that have been induced to turn back their developmental clock and lose markers of differentiation (to become stem cells), while regaining the ability to multiply). These cells are undifferentiated cells that have the capacity to develop into any of the different types of cells in the body but can also go on multiplying indefinitely to maintain the pool of undifferentiated cells. Once a cell differentiates into a specific type of cell, it can no longer change its direction of development, and in most cases, it cannot multiply any more.

The problem the researchers run into is that, unlike differentiated cells, stem cells aren’t responsive to the conventional CRISPR gene editing technology, though the reasons are not yet clear. To overcome this, the ASU team has adopted a modification to this technique, first reported by David Liu’s laboratory at Harvard, to edit genes in stem cells.

The study

The current study by David Brafman and his colleagues at Arizona State University (ASU) is focused on learning more about diseases like Alzheimer’s disease (AD) which are the result of degeneration in the central nervous system. The research is centred on the use of stem cells to study the effects of specific mutations or risk factors in this condition.

The current study uses a new reporting method called TREE (Transient Reporter for Editing Enrichment) to modify CRISPR, thus making it possible to enrich cell populations – and even human stem cells, for the first time - where the DNA base has been edited, at high efficiency. The whole difference between CRISPR and the modified TREE approach is that whereas the former makes a nick in both strands of DNA at once, the latter cuts only a single strand – and reports that it has done so. For instance, after changing a single DNA base from cytosine to thymine, a protein signals the change by changing color from blue to green.

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.”