Dr. Rebecca Roberts from Synthego has recently published an article discussing applications of CRISPR in cancer cell lines and how CRISPR editing is advancing cancer cell line research. The advent of CRISPR genome engineering technology has enabled many breakthroughs in the field of oncology.
The author has looked into some of the key examples of CRISPR cancer cell lines and how they have assisted with drug discovery, generated treatments for drug-resistant cancers, cell and gene therapies, and immuno-oncology. Below are several highlights from the article:
1.Drug discovery in cancer – using CRISPR to perform gene knockout
Like the field of infectious diseases, a critical barrier to curing cancer is drug resistance (see related publication 1). Many types of tumours cannot be cured, even when treated with complex regimens of surgery, radiotherapy, and polychemotherapy. As such, the focus of much cancer treatment research has recently shifted toward identifying and disrupting the mechanisms which allow normal cells to become cancerous and enable resistance to treatment.
In lung cancer, for example, drug resistance has been correlated with the upregulation of certain genes, including Nuclear Factor Erythroid 2-Related Factor (NRF2). NRF2 is responsible for the regulation of several hundred other genes, which are involved in cellular stress responses. Using CRISPR, a recent study performed knockouts of NRF2 (see related publication 2) in the A549 lung cancer cell line, resulting in reduced proliferation of cells and increased sensitivity to chemotherapy treatment. The study confirmed the results of the cell line experiments in a xenograft mouse model, where NRF2 knockout cells displayed significantly reduced rates of proliferation – even in the absence of chemotherapy treatment.
2. Precision disease modeling – using CRISPR to create complex cancer models
While cancer cell lines are useful, CRISPR has served to make them even more advantageous. Since cancer genomes are incredibly complex and vary between patients, precision models are a necessity to understand tumour biology and develop more effective treatments.
A. Engineered human organoids
Complex models bearing all the necessary mutations, deletions, and chromosomal abnormalities can now be generated fairly rapidly and efficiently using CRISPR. Examples include engineering human intestinal organoids to create models of colorectal cancer (see related publication 3) and acute models of myeloid leukemia (see related publication 4) generated from mouse hematopoietic stem cells (HSCs).
B. Confirm causative roles of a gene, identify genes essential for the growth of cancer cell lines, and gene knock-in
CRISPR can confirm causative roles for genetic mutations in genes of interest, such as oncogenes and tumor suppressor genes. It is also frequently used to identify genes that are essential for the growth and survival of cancer cell lines (see related publication 5) , like PANC-1, which is a model for pancreatic cancers. In the breast cancer cell line MCF7, CRISPR has been used to knock in functional copies of the FOXA1 (see related publication 6) transcription factor gene.
C. Create drug-resistant cellular models
Creating drug-resistant cellular models is also critical in cancer research but is typically a painstakingly slow and laborious process with low efficiencies. This is an area in which CRISPR has enabled significant and rapid advances, reducing timescales from years to weeks. In a 2018 study, researchers used CRISPR to generate knockouts of the mediator complex subunit 12 (MED12) gene in multiple cancer cell lines, creating resistant models for drug screening (see related publication 7) . These models can be used to determine the efficacy of anti-cancer drugs and combinations of these for treating drug-resistant tumours.
D. Generate specific mutations in the gene in cancer cell line
CRISPR has also been used to generate specific mutations in the epidermal growth factor receptor (EGFR) gene in the PC9 lung cancer cell line (see related publication 8) . The mutation in question, T790M, is known to cause resistance to tyrosine kinase inhibitors, which are usually effective treatment options for lung adenocarcinomas. Creating CRISPR-edited cancer cell lines like this allows researchers to investigate both drug efficacy and the mechanisms of drug resistance in mutated cells.
E. Generate cancer-related chromosomal aberrations in cell lines
Some cancers, such as acute myeloid leukemia and Ewing’s sarcoma, are caused by chromosomal translocations. Despite the clear need to study these in a disease model to generate more effective treatments, they have been difficult to recapitulate. CRISPR has now enabled these cancer-related chromosomal aberrations to be generated in nearly any cell line (see related publication 9), allowing research on these types of cancers to progress rapidly.
3. CRISPR studies for understanding gene function in cancer
Examine the effects of gene knockouts on cancer cells
There have been many recent studies examining the effects of gene knockouts on cancer cells to identify potential targets for cell and gene therapy. In ovarian cancer research, the gene ONECUT Homeobox 2 (OC-2), a transcription factor, was found to be highly expressed in ovarian cancer tissues and cell lines and was implicated in tumour progression. A recent study used CRISPR-Cas9 to knockout OC-2used CRISPR-Cas9 to knockout OC-2 (see related publication 10) in SKOV3 and CAO3 cells, leading to downregulation of several pro-angiogenic growth factors, suppression of tumor growth, and apoptosis.
Mutations in the proto-oncogene Kras have been detected in 90% of pancreatic ductal adenocarcinoma cases, making it another key target for CRISPR-based cancer treatment. A 2019 study used CRISPR-Cas9 to knock out heterozygous Kras mutations (see related publication 11) in three different cell lines, followed by an analysis of changes in signal transduction proteins, with the knockout cells resembling wild-type cells in terms of expression of signal transduction genes. This study both confirms Kras can be targeted to restore normal cell function and that specific knockdowns of Kras can be performed using CRISPR.
Progress has also been made toward finding a cure for cervical cancer using CRISPR-Cas9. Human papillomavirus (HPV) causes many cases of cervical cancer via degradation of p53, a tumour suppressor gene, by the HPV E6 oncoprotein. In a key study, CRISPR-Cas9 was used to perform knockouts of E6 in three HPV-positive cervical cancer cell lines (see related publication 12) . Subsequent analysis of genetic mutations, mutation frequency, protein expression levels, apoptosis, and cell proliferation revealed downregulation of E6, upregulation of p53, increased levels of apoptosis, and dose-dependent suppression of cell growth. An in the Vivo mouse model was then used to explore the effects of treatment in subcutaneous tumours – intratumoral administration resulted in significant suppression of tumour growth with no adverse events detected.
Other studies have used CRISPR with different Cas nucleases to provide potential gene therapies for cancer. A notable example is a 2020 paper that used CRISPR-Cas13a to knock out endogenous oncogenes such as TERT in the liver cancer cell line HepG2 (see related publication 13). As a result, the cell line displayed significant apoptosis and reduced growth, indicating that CRISPR editing of TERT and other oncogenes can be used as a treatment for liver cancer.
4. Immuno-oncology research – using CRISPR to develop universal CAR T cells for patients
One of the most significant recent advances in cancer treatment research has been in CAR T cell therapy. This involves collecting the T cells of a cancer patient, activating, and engineering them, using a retroviral vector, to express chimeric antigen receptors that are specific to antigens from their tumour cells. When expanded and re-infused back into the patient as CAR T cells, they will identify, attack, and eliminate cancerous cells. This is a highly patient-specific treatment, however, and to overcome this limitation, CRISPR is now being used to develop universal CAR T cells that can be used for any patient.
5. Plasmids Demonstrate Variable CRISPR Editing Efficiencies
Plasmids may also have a quality problem. When CRISPR components are transfected as plasmid DNA, transcription (of gRNA and Cas9) and translation (of Cas9) need to occur within the cell before any genome editing occurs. Not only does this extend experimental timelines, but it also risks discrepancies in gRNA expression or the assembly and folding of Cas9. Working with RNPs, on the other hand, avoids these issues because the sgRNA and protein components are made before transfection. The quality of the gRNA and Cas9 can be verified before they are introduced to cells.
Another benefit of the RNP format is that the gRNA is protected by Cas9 such that it is less prone to degradation. Furthermore, synthetically-made gRNA can be chemically modified to prevent endonuclease degradation and immune responses. Both of these factors enable the gRNA to target genomic DNA more efficiently during the short time it is active within the cell (Hendel & Bak et al. 2015).
Given the many benefits of RNPs, it is not surprising that they have consistently yielded high editing efficiencies across a variety of immortalized cells, primary cells, and stem cells (e.g., Hendel & Bak et al. 2015, Kim et al. 2014, Liang et al. 2015, Lin et al. 2014). “Knocking-in” a genetic sequence via homology-directed repair, a notoriously inefficient process, also appears to be enhanced by the use of RNPs (Gaj et al. 2017, Lin et al. 2014, Schumann et al. 2015).
CRISPR Editing is Advancing Cancer Cell Line Research
Dr. Roberts has also discussed the future of CRISPR editing in the article. Below are some highlights:
- CRISPR gene editing in human subjects is still in its infancy, and researchers have proceeded cautiously when it comes to clinical trials for gene therapies that could potentially cure cancer. However, CRISPR has many other applications that will contribute to cancer research.
- There are many hurdles throughout the process of clinical development for potential cancer treatments and cures, often because immortalized cell lines used in pre-clinical research do not always accurately reflect the biological complexity of disease in human subjects. Since CRISPR can produce complex edits in so many different types of cells, immortalized CRISPR cancer cell lines will become increasingly relevant models of disease in the coming years, likely accelerating the process of clinical trials and minimizing trial failures.
- Advances are constantly being made in the realm of CRISPR technology, including the discovery and engineering of new Cas nucleases, tailoring of methods to reduce off-target effects of gene editing, and the optimization of CRISPR delivery methods. These improvements will make it safer and more feasible to apply CRISPR directly to human subjects to cure cancer and other diseases.
- The next few years will no doubt see an increase in the number of clinical trials using CRISPR to treat various cancers, including by engineering immune cells to make them better able to detect and kill cancerous cells. In addition, high-throughput CRISPR screens have enabled the identification of many genes which may contribute to oncogenesis, which will, in turn, lead to novel treatments for these cancers.
Full article from Dr. Rebecca Roberts is available here:
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