The role of BRCA2 in cancer development : simulating a knockout
In 2022, just last year, 1.9 million people just in the US were diagnosed with cancer. Currently, the prevalence of cancer ranges from approximately 5.5% of the population.
Right now, scientists are battling this devastating disease that affects millions worldwide. Genetic mutations are one of the most significant risk factors for developing cancer. There are many different genetic mutations that are involved with cancer development. Some well-known ones are BRCA2/BRCA1, TP53, APC, and BCR-ABL.
But, how exactly do genetic mutations directly influence cancer development?
Genes that encourage the cells to multiply (oncogenes) regulate cell growth, differentiation, proliferation, etc. So, mutations in genes that control cell growth and division can alter their function. Disruption of their function can lead to uncontrolled cell growth and the formation of tumors.
However, that is just one reason, additionally:
- Mutations could affect genes that regulate apoptosis, cell death. Since this process involves an array of genes, mutations can result in the failure of the cell’s death, especially with transformed cells. Hence, they can lead to tumor formation
- The DNA repair mechanisms that prevent the accumulation of mutations in cells could be mutated themselves. Mutations may have an impact on the DDR (DNA damage response) pathways. DDR pathways are in charge of spotting and fixing broken DNA. A failure in DNA repair caused by mutations in DDR-related genes may result in genomic instability, which raises the risk of cancer and other genetic illnesses.
Overall, mutations in oncogenes (promote cell growth) can result in inactive, overactive, or just uncontrolled cell growth and division.
Tumor Suppressors:
Another class of genes is tumor suppressors, which regulate cell growth and division. The knockout/loss of their function can lead to the development of cancer. This is because they prevent cancer by repairing DNA damage, promoting cell death (apoptosis) as required, and overall regulating cells. Hence, mutations of the tumor suppressor genes can lead to a reduction in their function; uncontrolled cell growth, reduced ability to repair DNA damage, and increased cell proliferation (TP53 and RB1 are what regulate the cell cycle, so a mutation could lead to tumor development), increased susceptibility to the environment (p53 protein is essential for DNA repair and aids in the start of apoptosis when it’s required to get rid of damaged cells. P53 can aid in preventing the replication of damaged DNA when cells are exposed to radiation or toxins and can encourage apoptosis in permanently damaged cells.)
The development of cancer can be influenced by a variety of factors, including mutations in tumor suppressor genes.
BRCA2:
Overall, BRCA mutations have to do with an increased risk of cancer development (breast and ovarian cancer), according to the National Cancer Institute. Preventing the development of cancer and producing proteins that repair damaged DNA is done by the BRCA1 and BRA2 genes (Breast Cancer Gene 1 and Breast Cancer Gene 2). These, are tumor suppressor genes because they “suppress tumors”; when they have mutations or changes, cancer can develop.
Every person has two copies of the BRCA gene, one inherited from each parent, just as they have two kidneys, each with a unique set of functions. This redundancy offers a failsafe mechanism for DNA damage repair and cancer prevention. The other copy of the BRCA gene can still offer some level of cancer protection even if one copy isn’t working properly.
One normal copy of the BRCA gene can still offer some degree of protection against cancer, despite the risk being higher than with two normal copies, much as a person can survive with only one functioning kidney. The risk of developing cancer can be considerably increased by deleterious mutations or variations in both copies of the BRCA gene.
However, harmful variants of these genes could be inherited from either parent. A harmful variant is where there is a change in the DNA sequence that could interfere with its function.
People who inherit harmful variants in one of these genes have increased risks of several cancers — most notably breast and ovarian cancer, but also several additional types of cancer. People who have inherited a harmful variant in BRCA1 and BRCA2 also tend to develop cancer at younger ages than people who do not have such a variant. Cells that don’t have any functioning BRCA genes due to a somatic alteration can grow out of control and lead the person to develop cancer.
My experiment:
Using Benchling- a cloud software development and research platform. It offers a set of molecular biology techniques, such as CRISPR editing, molecular cloning, protein engineering, and DNA sequence design and analysis.
(Brief mention: This was my first time using benchling, so my experiment with knocking out the gene might not be perfect, it is simply a simulation. Additionally, if you don’t have a lot of previous knowledge about CRISPR, I recommend you read my previous articles)
As mentioned previously, if both BRCA1 and BRCA2 genes were knocked out, the cells would lose their ability to repair DNA and increase cancer development. So, using Benchling I was able to simulate a CRISPR knockout of BRCA2.
So, by importing the DNA sequence of a Homo sapiens genome and importing the BRCA2 sequence. By using the Design CRISPR function, I could find one important exon where I wanted to create a knockout.
However, with BRCA2, there are 2 different exons that are most frequently mutated and associated with an increased risk of cancer. Exon 11, the biggest exon, contains the highest frequency of disease-causing mutations. Overall, the mutations in this specific exon have been shown to overall disrupt the function of the gene. Additionally, Exon 3 has been proven to have been associated with an increased risk of breast cancer as well. The process of CRISPR assembling exon 11 versus exon 3 is similar in that both involve using guide RNAs to target and induce a double-stranded break (DSB) in the DNA at a specific location. However, there are some differences in how these two exons may be targeted and the potential effects of their disruption. So, comparing the potential effects of CRISPR-mediated disruption of Exon 11 versus Exon 3 of the BRCA1 or BRCA2 genes could provide valuable insights into the functional consequences of specific mutations and the role of these genes in cancer development.
So, I performed a simulation of a knockout of Exon 11 and 3.
Exon 11:
Above, the linear map of the BRCA2 gene has a prominent exon (Exon 11) where I can perform my knockout. By creating the adequate target region, I needed to decide where I wanted to specifically place my knockout and cut position. Benchling gave me 259 (I have only put a few below as an example) options for where I could place my cut position with the given target region (32336265–32341196).
But, how could I decide which one to pick; where I wanted my cut position? I could do this by looking at the On-Target-Score. The On-Target-Score is the percentage of the chance it will actually reach that target. Hence, the higher the score, the better. By filtering the scores from highest to lowest, I assembled the one with the highest. To assemble the CRISPR, I needed a Vector Source, a plasmid: lentiCRISPRv2.
You might not know what that means, but let me explain.
LentiCRISPRv2:
For CRISPR-Cas9 gene editing, there are many components needed for delivery into mammalian cells. One is, a plasmid with a promoter to drive the expression of the components, along with a gRNA. The guide RNA is what directs the Cas9 protein to the target site in the genome. Overall, a plasmid vector is a DNA molecule used to put DNA segments that are foreign into a host cell. In molecular biology, usually they are used in host cells to introduce or modify new or previously existing genes.
Specifically, LentiCRISPRv2 is a plasmid from a virus: lentivirus. This is what allows CRISPR-Cas9 components to be delivered into mammalian cell types. It is a very commonly used plasmid used in molecular biology: gene knockouts, gene repression, and gene activation.
… Back to the experiment…
After doing that, I was able to assemble the knockout by genetically manipulating the specific gene in the genome. So, after determining the target gene and generating the CRISPR-Cas9 system, the Cas9 nuclease and gRNA were able to be delivered into the cells using the plasmid vector. Hence, they targeted and cleaved the DNA at a specific location.
However, it doesn’t just stop there, we need to also perform a simulation of Exon 3, so by repeating the steps I did above with a different exon, I got the following:
Exon 3:
If I was actually doing this in a lab setting, and not just making a simulation, I could be able to verify the knockout through screening to make sure that the target gene was disrupted or deleted. Using methods like PCR, sequencing, or other, I would detect the changes in the DNA.
Rather watch a video of me explaining this? Watch here.
My sources:
