Description

Introduction

According to statistics from CA Cancer J Clin, breast cancer is the cancer with the highest morbidity and mortality among women ^[1]. Most deaths from breast cancer are associated with late detection, which causes patients missing out the best treatment period ^[2]. However, traditional breast cancer detection method like invasive tissue biopsy is often accompanied by pain of patients.

Therefore, our team wants to create BreFast -- a fast and convenient breast cancer screening kit. We hope that BreFast will improve the efficiency of breast cancer detection, so that breast cancer patients can receive treatment in time.

Meaning of "BreFast" & Our Logo

"BreFast" is formed by the combination of two words "breast" and "fast".

Breast Cancer Screening

Currently, the treatments of breast cancer mainly include surgery, radiotherapy, chemotherapy and targeted therapy ^[2]. These treatments are usually very effective for early-stage breast cancer patients. However for late-stage patients, these treatments cannot always achieve desirable results. About 30% of breast cancer patients die of cancer metastasis ^[3].

Many medical evidences demonstrate that breast cancer screening can improve early diagnosis of breast cancer, which effectively lower mortality rate of breast cancer patients ^[4]. The results of several prospective RCT clinical trials also shows that effective screening can lower the mortality rate of breast cancer by 20% ^[5].

Breast cancer screening aims to discover cancerous cells for early interference of cancer progression. In short term, breast cancer screening may lead to a sudden increase of breast cancer case as more hidden cases are discovered. Yet in a long run, the patient’s survival period is patently extended ^[6]. Additionally, early detection of breast cancer through screening can significantly improve patient outcomes. It increases the likelihood of preserving the patient's breasts, accelerates the recovery process, reduces the probability of complications, and helps prevent mental health issues associated with the disease ^[7]. What's more, the earlier the stage of breast cancer is discovered, the lower the probability of patients receiving chemotherapy is. Consequently, patients do not have to face the side effects of chemotherapy ranging from cardiotoxicity to myelotoxicity.

Figure 1 Ten leading cancer types for the estimated new cancer cases and deaths by sex, United States, 2023 ^[1].

miRNAs

MicroRNA (miRNA) is a small, single-stranded non-coding RNA molecule containing 21~23 nucleotides^[8], which can be found both inside cells and in the blood. MicroRNA binds to a complementary sequence in the mRNA to regulate gene expression and modulate cell function^[9,10].

Figure 2 MicroRNA binds to a complementary sequence in the mRNA to regulate gene expression and modulate cell function.

These days, circulating miRNA has become a popular target molecule to influence cell differentiation and a promising biomarker foor cancer diagnosis ^[11]. Through bioinformatics analysis, we identified a breast cancer-associated miRNA, miR-21-5p, as our biomarker. See the Bioinformatics Analysis page for details.

Bioinformatics Analysis

CRISPR/Cas13a system

CRISPR is an immune system of prokaryotes, including bacteria and archaea^[12]. These prokaryotes contain DNA sequences originating from previously infected bacteriophages, and RNA transcribed from these DNA sequences can be used as guide RNA to detect bacteriophages that have the same DNA sequences. Specifically, such guide RNA can be bound by Cas protein, and forms a RNA-DNA double strand with bacteriophage DNA that contains a complementary sequence, which subsequently activates the nuclease activity of Cas protein to cleave the bacteriophage DNA ^[12].

Figure 3 Working principle of CRISPR/Cas13a system^[13].

CRISPR/Cas13a is engineered as a RNA-guided RNA cleavage system ^[14]. Cas13a identifies loop region of crRNA, and forms Cas13a/crRNA complex. crRNA interacts with its target RNA that contains complementary sequence by forming double-strand RNA, which activates the nuclease activity of Cas13a. Activated Cas13a/crRNA complex cleaves not only the target RNA, but also any by-stander RNAs regardless of their sequence complementarity with crRNA ^[15].

We engineered a CRISPR/Cas13a system and tested its cleavage activity. See the Engineering and Results Page for details.

Engineering Results

Lateral Flow Detection

Lateral flow detection is a rapid test based on the specific binding between antigens and antibodies. It is simple, convenient and economical. Result is usually shown within 30 minutes ^[16].

In our project, lateral flow strip is designed to detect the cleaved reporter RNA. The main structures of the lateral flow strip include sample pad, probe pad, nitrocellulose membrane and absorbent pad. Test line (T line) and control line (C line) are for result displaying.

Figure 4 Diagram of lateral flow strip.

The working principle of the strip is shown in Figure 5. When a sample is added at sample pad, the sample flows along the strip, which passes through the probe pad to the test zone and finally to the absorbent pad. In the case that the reporter is uncleaved, gold-labeled streptavidin stored on the probe pad will bind to the biotin side of reporter and continue to migrate along the strip. Then, anti-FAM antibody situated at T line captures the FAM side of reporter, leading to accumulation of colloidal gold (shown in red). In the case that the reporter is cleaved, the biotin side is separated from the FAM side. As a result, anti-FAM antibody at T line cannot capture the biotin and the colloidal gold that binds to the biotin anymore. Thus, according to the presence of colloidal gold at T line, it is easy to know whether the reporter is cleaved or not. Besides, The sheep anti-mouse antibody on the quality Control line (line C) can capture the gold-labeled mouse antibody, which can prove that the sample flows through the entire strip. For more information, please click the Results Page.

Results

Figure 5 Design and principle of lateral flow strip.

How can BreFast be used to screen breast cancer?

Our solution

A fast and convenient breast cancer screening kit -- BreFast.

BreFast employs an engineered crRNA to specifically target miR-21-5p, a miRNA associated with breast cancer. Upon binding of the crRNA to the targeted miRNA, the nuclease activity of Cas13a is activated, leading to the cleavage of the reporter RNA. The cleaved reporter RNA can then be detected using a lateral flow strip. The relative abundance of cleaved reporter RNA detected on the strip correlates with the expression level of the miR-21-5p in the samples. Given its capability to detect breast cancer-associated miRNA, BreFast holds promise as a diagnostic tool for breast cancer screening.

Future application

In the case that BreFast will be used in actual breast cancer screening, it will include the following steps. Firstly, samples will be mixed with the Cas13a/crRNA complex. If a sample contains miR-21-5p, it will lead to the cleavage of the reporter RNA. Next, drops of the Cas13a reaction mixture will be added onto lateral flow strip. Finally, two bands on the strip indicate negative, while one band on the strip indicates positive for miR-21-5p. A positive result implies a high risk of breast cancer, and individuals with positive results will be suggested to go to hospital for further diagnosis to confirm whether they have breast cancer or not.

Figure 6 Diagram of the potential application of BreFast.

Advantages of BreFast

Many traditional miRNA testing techniques are based on qRT-PCR, which is time-consuming, expensive and requires reliable experimental skills. By comparison, BreFast takes advantages of the high sensitivity of CRISPR/Cas13a system and the convenience of lateral flow strips, making it easy to conduct breast cancer screening.

References

[1] Siegel, R. L., et al. (2023). "Cancer statistics, 2023." CA Cancer J Clin 73(1): 17-48.

[2] Jokar, N., et al. (2021). "Theranostic Approach in Breast Cancer: A Treasured Tailor for Future Oncology." Clin Nucl Med 46(8): e410-e420.

[3] Steeg, P. S. (2016). "Targeting metastasis." Nat Rev Cancer 16(4): 201-218.

[4] Mann, R. M., et al. (2020). "Novel Approaches to Screening for Breast Cancer." Radiology 297(2): 266-285.

[5] Oeffinger, K. C., et al. (2015). "Breast Cancer Screening for Women at Average Risk: 2015 Guideline Update From the American Cancer Society." Jama 314(15): 1599-1614.

[6] Marmot, M. G., et al. (2013). "The benefits and harms of breast cancer screening: an independent review." Br J Cancer 108(11): 2205-2240.

[7] Collins, K. K., et al. (2011). "Effects of breast cancer surgery and surgical side effects on body image over time." Breast Cancer Res Treat 126(1): 167-176.

[8] Green, D., et al. (2016). "Microguards and micromessengers of the genome." Heredity (Edinb) 116(2): 125-134.

[9] Schneider, M. R. (2012). "MicroRNAs as novel players in skin development, homeostasis and disease." Br J Dermatol 166(1): 22-28.

[10] Sengupta, D., et al. (2021). "Dissecting miRNA facilitated physiology and function in human breast cancer for therapeutic intervention." Semin Cancer Biol 72: 46-64.

[11] Xu, J., et al. (2020). "Roles of miRNA and lncRNA in triple-negative breast cancer." J Zhejiang Univ Sci B 21(9): 673-689.

[12] Koonin, E. V. and K. S. Makarova (2019). "Origins and evolution of CRISPR-Cas systems." Philos Trans R Soc Lond B Biol Sci 374(1772): 20180087.

[13] Shan, Y., et al. (2019). "High-Fidelity and Rapid Quantification of miRNA Combining crRNA Programmability and CRISPR/Cas13a trans-Cleavage Activity." Anal Chem 91(8): 5278-5285.

[14] Zhou, T., et al. (2020). "CRISPR/Cas13a Powered Portable Electrochemiluminescence Chip for Ultrasensitive and Specific MiRNA Detection." Adv Sci (Weinh) 7(13): 1903661.

[15] Kellner, M. J., et al. (2019). "SHERLOCK: nucleic acid detection with CRISPR nucleases." Nat Protoc 14(10): 2986-3012.

[16] Wang, Z., et al. (2021). "A strip of lateral flow gene assay using gold nanoparticles for point-of-care diagnosis of African swine fever virus in limited environment." Anal Bioanal Chem 413(18): 4665-4672.