Introduction

We collect information through public surveys, field trips, and expert interviews to guide our next work. Our project is about using CRISPR Cas12a cell-free system to target circRNA for early screening of breast cancer, which requires us to further our human practice work. Our main goal is to deeply understand the background knowledge of breast cancer, improve the technical route and improve the hardware device.

 

Highlights for Integrated Human Practices

 

Cas9àCas12a

In the beginning, we did relevant experiments using the CRISPR-Cas9 system, but the fluorescence signals were too low to be seen under ultraviolet light. As a result, we considered that the sensitivity of CRISPR-Cas 9 might be not that good, so we suspected that CRISPR-Cas9 was not specific enough in our cancer-related circRNA detection. Later on, according to Professor Sun's suggestion, we changed the CRISPR-Cas9 system to the CRISPR-Cas 12a system to ensure the specificity, sensitivity as well as the intensity of the fluorescence signals.

 

Better kit

To improve our experiment kit, we made our kit into a smaller, more convenient, and more accurate kit.

Since there are many components in our blood that we don't know about, they may be preventing our system from functioning properly more than we think. As a result, we visited xi 'an Tianbo medical testing laboratory, through the guidance of professional staff, we chose to centrifugal blood samples, extract the goal we want circRNA, abandoned yellow skin and red blood cells, can eliminate the influence of many unknown, and through the temperature preservation of blood sample, in order to keep the most accurate results.

In the actual use of the main need to pay attention to standardization and stability. It should be simple to operate, economical, with acceptable sensitivity and specificity. The accuracy of early diagnosis of breast cancer still depends on the experience of professionals to a large extent. Therefore, an accurate and convenient detection kit or equipment is still in great need. To address this issue, we may need to designate target audiences who have doubts about their breasts because they want to be examined before going to the hospital. 'We made the kits smaller, lighter and narrower so that users can better see the fluorescence,' said expert Hu Fei. Our kit can be used like a pregnancy test, which is both convenient and relatively accurate; They can go to the hospital if they want, but they can use our kit for pre-testing. In conclusion, we need to improve the accuracy and reputation of our project and let more people know about and use our toolkit.

 

 

An in-depth look of breast cancer

From the expert Dr. Jia and Dr. Yuan, we further understand breast cancer and the treatment device, which the cause of breast cancer can be multiple. Also, the importance of timely detection of early abnormal signals for treatment, and the importance of standardization and stability of methods

 

Purposeful and determined science communication

Purposeful and determined science communication summarizes the conclusions of expert interviews and questionnaires and identifies the objectives of science communication. We decided to inform the public about the dangers of breast cancer and raise public awareness of the scientific prevention of breast cancer. We also plan to give a brief introduction to our project to dispel any doubts about it. At the same time, we also plan to introduce the broad prospects of synthetic biology to the public and increase their awareness and attention to this field.

 

 

1 Background survey

According to the GLOBOCAN 2020 database, there were 2.3 million new breast cancer patients in 2020 surpassing lung cancer to be the most prevalent cancer in the world. Among all female cancers, breast cancer has both the top incidence (24.2%) and the top mortality (15.5%) in 2020 (Sung, 2021). Imaging, ultrasound, ultrasonography, mammography including nowadays digital mammography and tomography of mammography, and MRI are the common diagnostic tools for breast cancer. To create a more convenient diagnostic method, our team decided to create an early screening kit for breast cancer to further reduce the mortality rate in the future, and to increase awareness of this disease.

In the beginning, our team decided to use the CRISPR Cas9 system to determine our project because it is more commonly used and it is more mature. However, we chose the CRISPR Cas12a system in the final, because of the visibility of the fluorescent signal, and the accuracy of the experiment, and all these factors accomplished our projects with the CRISPR Cas12a system.

 

 

 

2 Public Survey 

Summary

From the survey results, we found that users have high acceptance and expectation of the early screening program, and a large proportion of respondents want to achieve self-testing at home due to the privacy involved in breast cancer. This helped us to initially identify the target users for our program. At the same time, the survey results also allowed us to further clarify the key points of our program design, such as accuracy, portability, low technical requirements, and low price.

Based on our research carried out on both underdeveloped area and developed local health system, we decide to build a breast cancer early detection kit which takes advantage of CRIPSR-Cas 12a system as a detection medium. And it can be used under different circumstances, providing a fast, accurate result. If our kit is used properly, it will relieve hundreds of millions of people in both underdeveloped region like Africa and developed countries like USA and China from breast cancer pain.

 

An online questionnaire was launched to find out how concerned people are about breast cancer and their habits of routine screening for breast health, as well as their views on current screening methods. 800 copies of questionnaires were collected and 1350 people viewed this questionnaire. Participants of different genders and diverse ages that have various educational backgrounds have taken part in the online questionnaire.

 

From the feedback of the questionnaire, it is indicated that a majority of 92.2% of the interviewers have heard of breast cancer and 45.9% of the respondents and the people around them had breast cancer. This further indicates that breast cancer has become a very common and highly prevalent disease in the public.

 

 

 

However, when referring to the specific clinical stage and symptoms for breast cancer, a majority of the public were not clear about it. As shown in the figure below, the public considered chest pain (72.6%), breast mass (87%), and nipple depression with abnormal secretion (60.6%) as the three major symptoms, while there are also some other symptoms to show breast cancer, and these symptoms have relatively fewer respondents considered to be serious symptoms that show breast cancer; and they are orange skin changes of the breast (44.5%), contraction of the breast tissue (41.3%), and chest pain, shortness of breath, pleural effusion, etc. (45.2%).

 

 

 

One of the best ways to prevent breast cancer is to do regular breast cancer screening. In accordance with the data, only 23.1% of the participants are currently in the habit of regular breast checks, and the remaining majority of 76.9% of the respondents don’t have the habit of regular breast cancer screening. Among them, the frequency of getting a breast cancer screening was mainly once per year (71.9%), and few of the respondents did it twice a year (17.8%). And only 46.60% of respondents often pay attention to and check their breasts. As we know, paying attention to the breast frequently could help to reduce the possibility of being too late in treating breast cancer.

 

 

In order to understand public attitudes towards breast cancer treatment and screening methods, we set up questions on "whether breast cancer is curable" and "whether you would accept early screening programs"，our questionnaire revealed that the majority of the group of our responders think that it can be cured early (71.5%) and 87.8% of the participants will attend a early screening program. These results also gave us full confidence to start our project

 

 

 

 

 

 

In order to make our program, and especially the final product, more in line with public expectations, we surveyed users' perceptions about the early screening program. The results showed that among the respondents who received the early screening program 69.5% of their expectations for this project are due to the convenience of testing, and it is best to conduct self-determination at home, 43.3% are due to low cost, and 75.8% are due to high detection accuracy.

 

Of the respondents who did not receive the early screening program, 67.4% of them rejected the project because it was unnecessary, I was healthy, 36.8% because they did not have time, 11.6% because they were afraid of poor examination results, and 13.7% because they were afraid that the fees were too expensive.

 

This allowed us to further clarify the key points of our program design, such as accuracy, portability, low technical requirements, and low price. From the survey results, we also found that users have high acceptance and expectation of the early screening program, and a large proportion of respondents want to achieve self-testing at home due to the privacy of breast cancer. This helped us to initially identify the target users for our program.

 

 

 

 

3 Expert Interviews and field trip

 

Jia Jia, Deputy Chief Physician of the Department of Oncology, Chinese People's Liberation Army General Hospital

Highlight:

• Further understanding of breast cancer and treatment device: the cause of breast cancer can be multiple. The importance of timely detection of early abnormal signals for treatment

• The importance of standardization and stability of methods

 

Before the interviews, we were aware of the number of breast cancer patients annually around the world and the detrimental impacts of breast cancer on people around the world. However, from the interviews, we were informed several times that the cause of breast cancer can be multiple. The high fatality rate is the result of the failure of paying attention to some early abnormal signals in the body, and some of them are already in the late stage when discovered, and now everyone lives at a fast pace, ignoring the importance of early regular inspections. Although people have paid more attention to breast cancer now, the general public's health awareness is not strong enough and needs to be further improved.

CRISPR technology has a promising future in breast cancer from diagnosis to treatment, but there is still a long way to go before it is popular. Because first, everyone feels that this technology is abstruse, but it is not the case.

 We are currently building a kit for breast cancer diagnosis using CRISPR technology, and the target to be detected is specific circular RNA. For this problem, Jia said the main thing is to focus on standardization and stability because if we do not have a common standard, it will lead to failures of detection or mistakes. CRISPR technology has great potential in the diagnosis and treatment of tumors, and research is done one by one.

 

 

Yuan Huiling, Director of Breast Surgery, Dongguan People's Hospital

Highlights:

An in-depth look at the current state of breast cancer

 

The current state of breast cancer is now a less serious situation, most people choose to have their breast cancer screening in the hospital, and cure their illnesses in the hospital. To understand more about the meaning of our experiment, we interviewed Ms. Yuan.

There are three different types of breast cancer which are type A, and type B, including triple negative. As time pasts, it will be meaningful if we can have an instrument to help detect these three kinds of breast cancer at the same time, which the kit that we are designing is meaningful.

Within the expectation, our project perfectly filled in the blank of needed early breast cancer screening. There are 3 types of breast cancer, and it will be complex for people to screen and identify which of them is that particular breast cancer. However, our project of designing a convenient early breast cancer screening kit, help to solve this problem, because the theory that we are using is the CRISPR-Cas12a system, and we are detecting two circRNAs, which helped to quickly identify the existence of cancer.

There are three suggestions that Ms. Yuan provided to us about the kit that we are designing. The first point is that we should perform it on a small sample at the beginning because it involves a very strict ethical examination of the information in the human body. The second point is to have the patient's consent. The third point is that you need to refine your testing methods and refine your equipment.

 

 

 

Dongchang Sun, Associate Professor, Department of Bioengineering, College of Bioengineering, Zhejiang University of Technology.

Highlight:

• Adjust our project design: select cas12a system to replace cas9

 

Since the cas9 system we designed at the beginning did not work in the actual initial tests and could not achieve our goal of detection, we interviewed Professor Sun, an expert in gene editing. Changing from Cas9 to the Cas12a system could improve accuracy, avoid false positives, and increase the visibility of fluorescent light. The theory of the Cas9 system identifies double-strand and cut double-strand, although the CRISPR-Cas9 technique is highly precise in theory but is not in actual use. It can cause "off-target" changes, which are mutations in the genome that are not intended. Plus, the fluorescent light won't be as obvious as the Cas12a system because of it. In contrast, the theory of the Cas12a system will be more suitable for your project, since your team is using circRNA; it will be easier to apply the Cas12a system, the identifies double-strand and cur single-strand. The CRISPR Cas12a system could easily cut the fam group because it is more sensitive and flexible.

 

Fei Hu, associate researcher, Deputy director of the secretariat of the Key Laboratory of Pharmaceutical Microbiology Testing Technology of the State Drug Administration.

Research field: Medical-industrial cross bioassay medical device and precision instrument manufacturing

Highlight:

• Improved the design of CRISPR-Cas12a system and the actual kit

 

To succeed in our designed kit, we first need to understand the system more in-depth. We designed a kit for early breast cancer screening by using the CRISPR-Cas12a system, and the proposed target is circRNA. To improve our kit, we interviewed a specialist of instrument manufacturing, and he gave us some constructive suggestions. To make the kit more effective is to design an effective crRNA sequence, which is limited by the PAM site. In actual use, the most important thing to pay attention to is the processing steps before the sample is added to the detection system. Since blood contains a lot of complex components, therefore, our team need to understand how to deal with the real blood samples we get before we use the CAS12a system to test it. We can’t be sure if there are components in the blood that may prevent the CAS12 system from responding. We can do some more experiments to test the hypothesis. As for the detection part of the kit, in addition to ultraviolet light, we can also see fluorescence with blue and green light. We can make the box smaller, and lighter, make it narrower, so that the users can see the fluorescence better.

 

 

 

 

 

 

Field trip to Xi'an Tianbo Medical Testing Laboratory

Highlights: 

Instruction for our test kits is needed to guide the users how to use our kits.

 

In order to have a more detailed comprehension of breast cancer testing methods and the improvements of our experience kit, we attend the medical testing laboratory as a field trip on the 1st of September.

 

A staff member showed our team members around the company and introduced the functioning treating machines and various biological treatment strategy tools. We interviewed the staff member and asked for opinions to improve our project. The staff member has given valuable pieces of advice on our kit and specific advice on treating the blood sample. For instance, we should carefully label how should different types of people get the sample by themselves. For elders, children, and people facing disorders, the staff's suggestion is to emphasize in the instruction book to clarify the necessity of supervision from professional doctors, because to collect the blood we need for the kit, we need to collect it from different parts of the body, for adults, we normally utilize the median cubital vein or basilic vein on the arms. For kids, we get the blood sample from the jugular vein or from the scalp since their blood vessels are too thin. And for the diagnostic method, they suggested we use a serum to detect circRNA and to extract the circRNA we will need a centrifuge. Also, to ensure the accuracy of the test make sure to preserve them at 2-8 Celsius degrees after you centrifuge them and get the serum, and freeze the samples at -76 -- -80 Celsius degrees after the detection is done. Plus, the staff recommends we make a fluorescence intensity gradient to include in our instruction book so everyone can contrast the intensity of the fluorescence and is able to make the correct decision.

 

 

References

 Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA: A Cancer Journal for Clinicians, 71:209–249(2021).