Nature Medicine conducted two CRISPR related articles on Monday. Both studies found that the CRISPR-Cas9 system can induce intracellular p53-mediated DNA damage and affect CRISPR genome editing.
CRISPR-related companies’ share prices dropped sharply after the article went live, CRISPR Therapeutics fell nearly 13%, Editas Medicine fell nearly 8%, Intellia Therapeutics fell nearly 10%, and Sangamo Therapeutics fell nearly 5%.
The reason why investors react so strongly is that many people are worried that the risk of cancerous cells edited by CRISPR will increase. But, is this the truth? Let’s take a look at the research content of these two articles.
Interpretation of the article
The researchers in these two articles were from the Karolinska Institute and Novartis. Researchers at Novartis and Karolinska used different types of cells, respectively, and Karolinska’s lab uses a retinal pigment epithelial cell (RPE1). The RPE1 used in their experiments does not belong to the transformed cell, but its telomerase reverse transcriptase can stably express, thus inhibiting telomerase activity and immortalizing it. These cells are derived from human retinal pigment epithelial cells and have a high correlation with the development of CRISPR-related therapies for the treatment of retinal degenerative diseases.
The discovery of Karolinska Laboratories is also an accident. When they performed dropout screens on multiple cell lines using the CRISPR–Cas9 system, they found that the guide sequences targeting important genes in RPE1 cells were not effectively removed.
However, they also found a significant increase in the concentration of guides targeting the TP53 gene, which encodes p53. The authors believe that this phenomenon may be due to Cas9-induced double-strand DNA break (DSB) can activate p53, resulting in cell growth inhibition.
The laboratory also confirmed the above inferences through a series of experiments: If Cas9 is used to edit the genome in cells with normal p53 function, it can initiate a DNA damage response and lead to cell cycle arrest.
However, we can also see from the article that this phenomenon depends on the existence of DSB. If you use the CRISPR system without shear function to edit the genome, it may not produce this phenomenon.
The study also found that if the DNA damage response is inhibited, the precise editing efficiency of CRISPR in normal non-transformed cells can be improved, but this operation will also allow the damaged genome to escape the intracellular DNA damage detection system.
Compared to Karolinska’s article, Novartis’s article has a higher degree of concern (or is it more topical?). Novartis used human pluripotent stem cells in this study. Since such cells can differentiate to form almost any type of cell, they are also likely to be used for the treatment of many types of diseases.
However, many previous studies have found that gene targeting in human pluripotent stem cells is much less efficient than other types of cells. So Novartis’s team hopes to increase the efficiency of CRISPR editing in pluripotent stem cells.
Novartis’s team has increased the efficiency of Indels in pluripotent stem cells to 80% through the All in one inducible Cas9 + doxycycline system. But they also found that only a small fraction of cells survived after genetic editing. And, unfortunately, they finally discovered that CRISPR could successfully edit these cells because the lack of p53 function in these cells enabled the genome editor to proceed smoothly.
In fact, this is also very easy to understand, because pluripotent stem cells are very similar to cells in early embryos, and extreme sensitivity to DSB can inhibit the further proliferation of abnormal cells in the embryo to some extent, which is also a self-protection mechanism.
The function of p53 in these two articles is somewhat different. In Karolinska’s article, DNA double strand breaks can activate p53, leading to cell cycle arrest. However, in Novartis’s article, the impact of p53 is much more serious, because they found that many of the cells that could successfully edit were those with defects in p53 function. Cells without p53 deficiency did not have normal survival.
In other words, the CRISPR-Cas9 system used in this experiment can screen out those cells that have defects in p53 function. Since p53 is an anti-oncogene, applying these cells with defects in p53 function to disease treatment may result in The formation of tumors.
How big is the impact?
I think the impact of these two studies is actually not as large as most investors think. Because even if p53 affects the genome editing of the CRISPR-Cas9 system in certain types of cells, it does not mean that all CRISPR therapeutics have this problem.
First, this problem does not exist in genome editing where there is no double stranded DNA breakage at least for the time being. For example, Beam Therapeutics, a joint venture between Zhang Feng, David Liu, and J Keith Joung last month, is hard to be affected. Because the company uses CRISPR to edit bases, it does not involve DNA cleavage and fragmentation.
Besides base editing, the CRISPR-Cas9 system using the NHEJ mechanism may not be affected. CRISPR therapy for disease treatment mainly edits DNA sequence in two ways, one is to use the non homologous terminal connection (NHEJ) mechanism to form indel, which can destroy the function of the original gene, which is also a strategy for the treatment of sickle cell anemia. This strategy is also called gene disruption. In addition to this mechanism, homologous mediated repair (HDR) can also be introduced to other genes. This strategy is called gene correction.
Emma Haapaniemi, the first author of Karolinska’s article, told Stat News that CRISPR-Cas9, based on the NHEJ repair mechanism in their experiments, still performs normal genome editing even when p53 function is activated.
As a result, R&D projects based on the NHEJ mechanism in the pipelines of companies such as CRISPR Therapeutics and Editas Medicine may not be affected by p53. However, some HDR-related research projects may be affected by these studies. As far as the impact is concerned, we need to conduct follow-up research before we can know.
CRISPR Therapeutics and Editas Medicine’s pipeline
It should be emphasized that these studies are only some of the earlier research data. Whether the cells used in the relevant company studies will be interfered by p53 during the CRISPR-Cas9 editing process, resulting in the enrichment of p53-deficient cells. It is still unknown.
And I think that the risk of cancer that people are worried about is probably only relatively easy to find in the study of pluripotent stem cells. Because this is largely due to the characteristics of the pluripotent stem cells themselves.
The proportion of p53 mutations that are commonly used in induced pluripotent stem cells is particularly high, reaching 3.5%, and the pluripotent stem cells frequently used in transcriptome sequencing have a mutation rate as high as 29%. In combination with this type of cell, the sensitivity to DNA damage response is very high, so it is not surprising that there is an enrichment of p53-deficient cells after gene editing in these types of cells.
In fact, as early as July last year, the preprint version of Novartis had already gone live on bioRxiv, but it did not cause much reaction. The reason why this week’s stock of CRISPR-related companies in the two articles fell collectively was probably due to the fact that the title of “causing cancer” used in media reports was too compelling.
In fact, this is not the first time that CRISPR has suffered a setback. In 2017, an article in Nature Methods reported that CRISPR could trigger a wide range of off-target effects, but this article was retracted in March this year. In January of this year, bioRxiv launched another article saying that the human body could have an immune response to Cas9. In the same way, the online launch of these articles can trigger the turbulence of the stock price of the company concerned.
However, whether it is off-target effect, p53, or immune response, these are not unexpected phenomena of scientists, and there is no corresponding solution. But obviously many investors do not understand this.
Loncar voted on Twitter last month and asked why he thinks CRISPR Therapeutics is the most valuable company in the CRISPR field. The answer for most people is that he has the word CRISPR in his name.
Although it sounds funny, I think a large part of investors actually do not understand what CRISPR is. Due to the misperceptions caused by media reports, many people have overestimated this technology. This may also be one of the reasons why the market value of these companies is currently so high.
Although CRISPR technology has had a revolutionary impact on basic scientific research since its inception in 2012, there is still a long way to go before it can be applied to the treatment of diseases.
Last year, an article on Plos One found through data analysis that it took nearly three decades from the emergence of new technology to the launch of related drugs, and that CRISPR technology has been in existence for only six years.
Even the CAR-T technology, which has been hot for the past two years, has been in existence for 20 to 30 years since its birth. One very important purpose of writing these articles is to remind everyone that it is more difficult to apply new technologies and new drugs to the clinical than many people think. CRISPR is no exception.