Team:CCU Taiwan/Overview
ASFAST system
ASFAST system is a double-stranded DNA (dsDNA) detection device, which combines CRISPR-Cas12a, PicoGreen, fluorometer and microcomputer to detect the viral genome (i.e. ASFV) from blood samples. In the ASFAST system, CRISPR-Cas12a is applied to specifically detect the DNA sequence of the viral capsid gene, p72. The presence of p72 DNA sequence will change the fluorescence intensity of PicoGreen, which is recorded by fluorometer and quantized by microcomputer.
The CRISPR-Cas system is a prokaryotic defence mechanism against foreign genetic elements, including double strand (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double strand RNA (dsRNA). Cas12a (also called Cpf1) has a high specificity for targeted dsDNA cleavage (cis-activity), which is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence. The cis-activity of Cas12a may transiently trigger single-stranded DNA (ssDNA) cleavage (trans activity). Both cis and trans target a single active site, called the RuvC domain. Notably, the Lachnospiraceae bacterium ND2006 (Lb) Cas12a stands out among the Cas12a orthologs for its trans nuclease activity.
Nowadays, fluorescence or fluorochrome are commonly applied to amplify signals in biomedical assays and bioanalytical techniques. PicoGreen is an intercalating fluorochrome that we chose as the medium to indicate the trans-activation of Cas12a in our ASFAST system. On binding to DNA, the intercalation and electrostatic interactions between DNA and PicoGreen resulted in a >1000-fold enhancement of its fluorescence. Therefore, we can use PicoGreen to get a quantitative measurement of the amplified signal to detect the existence of viral dsDNA. Based on the results of this study, a model for PicoGreen/DNA complex formation is proposed.
Our experimental design focuses mainly on manipulating the experimental process to prove the feasibility of our idea, develop the protocol and model the mechanism of CRISPR Cas12a’s cis-trans cleavage activity.
The CRISPR-Cas system is a prokaryotic defence mechanism against foreign genetic elements, including double strand (dsDNA), single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double strand RNA (dsRNA). Cas12a (also called Cpf1) has a high specificity for targeted dsDNA cleavage (cis-activity), which is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence. The cis-activity of Cas12a may transiently trigger single-stranded DNA (ssDNA) cleavage (trans activity). Both cis and trans target a single active site, called the RuvC domain. Notably, the Lachnospiraceae bacterium ND2006 (Lb) Cas12a stands out among the Cas12a orthologs for its trans nuclease activity.
Nowadays, fluorescence or fluorochrome are commonly applied to amplify signals in biomedical assays and bioanalytical techniques. PicoGreen is an intercalating fluorochrome that we chose as the medium to indicate the trans-activation of Cas12a in our ASFAST system. On binding to DNA, the intercalation and electrostatic interactions between DNA and PicoGreen resulted in a >1000-fold enhancement of its fluorescence. Therefore, we can use PicoGreen to get a quantitative measurement of the amplified signal to detect the existence of viral dsDNA. Based on the results of this study, a model for PicoGreen/DNA complex formation is proposed.
Our experimental design focuses mainly on manipulating the experimental process to prove the feasibility of our idea, develop the protocol and model the mechanism of CRISPR Cas12a’s cis-trans cleavage activity.
Portable testing device
Since transporting the blood samples from farm to the lab results in a high risk of virus spread, we assert that conducting the ASFV test on the spot will bring a significant advantage in preventing the spread of ASFV. Thus, we developed a portable testing device to provide on the spot ASFV tests on the farm. The device also features an automated test procedure to ensure that the entire test procedure is regulated, in order to provide accurate test results.
Figure 1. CAD illustration of device.
Software package
Our team is dedicated in creating a superior software package to drive the device, as it is crucial to the automated test procedure. As we have reached out to potential customers and government agencies, they also suggested that a data tracking system is necessary. That means, every test results must be synced and managed by the local government as soon as the results are known.
To accomplish these two objectives, we created a software package which includes a Linux program, an Android application, and a cloud database. The Linux program will control the entire test procedure and process the fluorescence data, while the Android application is built to interact with the operator and cloud database. It collects the identity information of test subject, displays the test results on an Android phone and uploads the results to an online database. These three programs are built across different platforms to carry out specific tasks. They are also connected via wireless network to share information.
To accomplish these two objectives, we created a software package which includes a Linux program, an Android application, and a cloud database. The Linux program will control the entire test procedure and process the fluorescence data, while the Android application is built to interact with the operator and cloud database. It collects the identity information of test subject, displays the test results on an Android phone and uploads the results to an online database. These three programs are built across different platforms to carry out specific tasks. They are also connected via wireless network to share information.
Kinetic Model
Some may worry about the false negatives in ASFAST system. To avoid this problem, our team created a kinetic model, which simulates all the reactions happening in ASFAST. The model describes how the concentration of virus DNA affects the required reaction time, so we can provide sufficient time to all the reactions to avoid false negatives. We also analyzed the simulations and set the detection limit of ASFAST based on the concentration of virus DNA and the required reaction time.
Market analysis
We intend to develop ASFAST as a portable, valuable and user-friendly device, therefore conducting market analysis is necessary for our product. After conducting a market analysis, we gained valuable insight and information on our specific market and we would like to sell a service instead of the device. Our data tracking system and data access limitation system which provide the advantage of allowing full cooperation between ASFAST and the government in preventing or controlling an outbreak of ASF, therefore our main customer will be the government.
Meanwhile, to get substantial information in order to develop ASFAST according to the demands of the market, we took initiative to consult experts, professors and those in related industries, conducted surveys and even collaborated with other iGEM teams around the world. We further conducted programs to transmit knowledge, about both synthetic biology and ASF, to raise the awareness of the public as well.
Meanwhile, to get substantial information in order to develop ASFAST according to the demands of the market, we took initiative to consult experts, professors and those in related industries, conducted surveys and even collaborated with other iGEM teams around the world. We further conducted programs to transmit knowledge, about both synthetic biology and ASF, to raise the awareness of the public as well.
Policy enforcement
Without a shadow of doubt, we are devoted to countering the danger of ASF with our hearts and souls. Nevertheless, finding the right antidote before taking any action is essential to creating a good solution. We became acquainted with the problem from various perspectives. Before pork appears on our plates, there is a chain in the pork industry from the pig farm to the market. We found that there are some loopholes in the procedures of the pork industry chain, therefore, we are devoted to implementing a “Routine Health Check System” frequently and integrating ASFV detection into the pig health statement. Most importantly, this idea requires the acknowledgment and support of The Council of Agriculture in Taiwan, so we have consulted with them about how to implement a proactive solution before ASFV reaches Taiwan.
- Chen, J. S., Ma, E., Harrington, L. B., Da Costa, M., Tian, X., Palefsky, J. M., & Doudna, J. A. (2018). CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity. Science 360, 436–439.
- Ormerod, M. G., & Novo, D. (2008). Flow cytometry: a basic introduction. Michael G. Ormerod.
- Bellini, S., Rutili, D., & Guberti, V. (2016). Preventive measures aimed at minimizing the risk of African swine fever virus spread in pig farming systems. Acta Veterinaria Scandinavica volume 58:82.