CRISPR-Cas9, the p53 Pathway and Cancer Treatment
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Cancer and p53
A change or damage in genes causes cancers. These changes are called mutations, which stop genes from functioning normally. According to Cancer Net, one of the most commonly mutated genes that is found in people with cancer is called the p53 gene (also known as TP53). The Cancer Institute says that this gene produces a protein located in the nucleus of cells and is essential in directing cell division and death. The p53 gene belongs to the tumour suppressor gene family.
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Short for clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9, Medline Plus states that the CRISPR-Cas9 system was derived from an existing gene editing mechanism in bacteria. The bacteria collect parts of DNA from viruses and utilise them to construct CRISPR arrays (DNA segments). This enables bacteria to remember the viruses. Thus, once the viruses strike again, the bacteria use the CRISPR arrays to generate RNA segments that will attack the viruses’ DNA, disabling the virus.
CRISPR-Cas9 Use in Laboratories
In the lab, researchers use the same mechanism as previously described. According to Medline Plus, they create a piece of RNA with a guide sequence that connects to a specific target sequence of DNA. This piece of RNA binds to the Cas9 enzyme, which cuts the DNA to the desired location. Other enzymes such as Cpf1 can be used as well. After cutting the DNA, the researchers employ the cell’s DNA repair mechanism to add or remove fragments of genetic information or modify the DNA by replacing an existing segment with another tailored DNA sequence.
CRISPR-Cas9 and p53
To begin, it is important to know the differences between some terms. In a research article by Neil T. Pfister and Carol Prives, Wild-type TP53 (TP53-WT) is a sequence-specific transcription factor that activates cellular outcomes, such as cell arrest and cell death when activated by various stresses, metabolic changes, and more, depending on the stress. On the other hand, an article on Intra-tumor heterogeneity states that TP53-null (TP53/) are a specific group of tumours characterized by nonsense, frameshift, or splice-site mutations correlated with a total absence of p53 expression.
In a study published in May 2020, research demonstrated that p53 the pathway was upregulated (more highly expressed) when Cas9 was introduced into 165 pairs of human cancer cell lines and their Cas9-expressing derivatives, according to gene expression profiling of wild-type TP53 (TP53-WT) cell lines. According to Current Biology, seeing as the p53 is a tumour suppressor protein, its activation stimulates the formation of a range of gene products, which result in either a prolonged cell-cycle arrest in the G1 phase of the cycle, thus inhibiting the proliferation of damaged cells, or apoptosis, which removes damaged cells from our bodies. This was shown to be true at both the messenger RNA (mRNA) and protein levels. Furthermore, Cas9-expressing cell lines showed increased amounts of DNA repair. The genetic analysis of 42 cell line pairings revealed that the injection of Cas9 could result in the appearance and spread of p53-inactivating mutations. Competition tests in isogenic (similar genotypes) TP53-WT and TP53-null (TP53/) cell lines verified this.
Finally, they observed that Cas9 activity was lower in TP53-WT cell lines than in TP53-mutant cell lines, and Cas9-induced p53 pathway activation altered cellular sensitivity to both genetic and pharmacological perturbations. These discoveries may have significant ramifications for the correct application of CRISPR–Cas9-mediated genome editing.
Science Signaling shows another study published in June of 2018, where p53 was shown to antagonize the efficiency of Cas9-mediated gene modification in target cells. It demonstrated that CRISPR-Cas9 editing worked best in p53-deficient cell cultures. Deleting or reducing p53 ended up enhancing the amount of surviving cells with an altered genome in immortalized human retinal pigment epithelial (hRPE) cells and human pluripotent stem cells (hPSCs). The CRISPR technology selects for cells with a deficiency in p53, which means that edited cells are more vulnerable and could result in tumours.
Image is Courtesy of eLife.
CRISPR and Cancer Treatment
According to Web MD, researchers have successfully shown that they can destroy cancer cells in mice using gene editing. According to their findings, lipid nanoparticles may be able to enter cancer cells and give instructions for the production of CRISPR enzymes, which can tear apart the genetic material of those cells and destroy them. However, they did face some problems in their research. Cells have a defense mechanism that protects their genetic material from any manipulation. Multiple researchers from different educational institutions such as New York University, University of Tel Aviv, Harvard University, and a company called Integrated DNA Technologies developed lipid particles capable of delivering the instructions used to make CRISPR-Cas9 enzymes into cells. The cells then can use said instructions to make the enzymes, which would cut the DNA of cancer cells and destroy them. There is, however, a specific point mentioned by Dan Peer, Ph.D., the director of the Laboratory of Precision NanoMedicine at Tel Aviv University: “We want to make sure that this payload is focused into the right cell. You don’t want to edit nearby healthy cells. You want to edit only the disease cells. What we call off-target effects are potentially very high. What we have shown, with our strategy, if you look at nearby cells, we found there’s no activity. There’s no editing.” This is limiting the scientists from going into the clinical trial on humans. Still, they do want to push this research to its limits and eventually start doing clinical trials to advance cancer care and therapies further as it seems to be an up-and-coming field for cancer treatments.
Article Author: Celine Guirguis
Article Editors: Edie Whittington, Maria Giroux