Overall Experiment Timeline
Experimental timeline of the entire project. This describes all the experiments that are planned to take place until the completion of the entire project.
We envision that our overall experimental timeline will be comprised of three important phases:
- We test our detection systems on purified DNA and narrow down our options to the final detection system.
- - This is to ensure that our detection systems is functional, but also to narrow down the best detection system.
- - We will be transforming E.coli cells with the Bd target gene: the ITS Region for Bd. Similarly, we will be transforming a different set of E.coli cells with the Bsal target gene: the ITS region for Bsal. Note: The ITS regions for Bd and Bsal are distinct.
- - Due to the pandemic, acquiring samples of *Batrachochytrium dendrobatidis* and *Batrachochytrium salamandrivorans* was difficult, so performing experiments on E.coli would not only be safer, but easier to obtain.
- - Firstly, we will lyse the fungal zoospores and purify its DNA to ensure our detection system is functional on zoospore DNA.
- - Secondly and the final step of experimentation, we will work with whole fungal zoospores with our prototype.
Amplification and Detection
The current standard mode of Bd and Bsal detection is qPCR. In our iGEM project, we introduce a novel detection technique by coupling an isothermal amplification reaction with CRISPR detection. Hypothetically, this coupled system will significantly increase the specificity of our detection technique. Subsequently, our engineering team will be integrating this detection technique into a field-ready point-of-care device that will detect the presence of the fungal infection within 30-60 minutes. The ultimate aim of our detection system is to identify the best combination of amplification and CRISPR/Cas enzymes in order to give the most sensitive and specific detection technique. In current literature, there are numerous amplification methods and two particular CRISPR enzymes - CRISPR/Cas12a and CRISPR/Cas13. Our brainstorming process included the necessary research and experimental planning for .
Amplification: RPA and LAMP
Our brainstorming process in amplification included a plethora of amplification techniques, each with their own strengths and weaknesses. Keeping in mind the identified problem of our project, we established key aspects of an amplification reaction that would be suitable to be integrated into a point-of-care diagnostics device:
- Highly sensitive
We narrowed down our choices into two amplification reactions: RPA and LAMP; both were identified to be highly sensitive, isothermal, and relatively quick.
Loop-mediated AMPlification (LAMP) is a technique that amplifies DNA at 65℃ in 20 minutes. It accomplishes this by having a different kind of DNA Polymerase, a strand-displacing polymerase, that is capable of separating the strands at a lower temperature and elongating the primers. However, LAMP requires a bit more complex set of primers, because the goal is to form terminal loops on the synthesized DNA. The use of 4 primers that specifically recognize 6 distinct regions on the target gene increases the specificity. When designing the primers, one has to make sure two of them match an exact sequence on the DNA, so that when they are extended, they can form a loop between the primer and the inverse sequence on the new DNA. This process is repeated until both ends have the loops. The new DNA now has 6 possible starting sequences for DNA replication versus 2 in PCR. The loop structure allows us to make complex hierarchical structures capable of amplifying these starting sites, leading to even faster DNA replication. (Notomi, 2000)
Recombinase Polymerase Amplification (RPA) is a technique that amplifies a small amount of DNA at 37℃ in 10-20 minutes. It runs at a lower constant temperature compared to other techniques such as PCR, which requires a thermocycler. It uses a special set of enzymes called Recombinases to insert the primers into homologous or similar sequences on the DNA, which helps start the separation of the strands. A special strand-displacing DNA polymerase then makes sure the primer is extended along the DNA to be amplified requiring no higher temperatures than 37℃. Because the temperature is so low, the separated strands of the DNA can easily rehybridize or re-bind, which will interfere with the DNA replication process. To make sure this does not happen, a family of proteins called single-strand-binding proteins, or SSB proteins, bind to the separated strands to prevent them from reforming hydrogen bonds. As the DNA polymerases extend the primers on both strands, the original DNA strands fully separate, eventually producing 2 new molecules of the same DNA. The steps are repeated for each new DNA molecule and the exponential amplification of just a small amount of starting DNA is achieved. (Lobato, 2017)
RPA vs. LAMP
In order to determine the best amplification reaction for our diagnostics device, we will test for two parameters in the lab: sensitivity and specificity. We determined these two parameters to be the required factors to establish the superior amplification technique.
To test for sensitivity, for each amplification reaction, we plan on conducting amplification reactions on serial dilutions of DNA concentrations. PCR will also be used on serial dilutions of DNA as a standard for comparison with RPA and LAMP. In each amplification reaction, we will compare the lowest DNA concentration each reaction will be able to amplify and rank the sensitivities accordingly. Our results will be visualized through SYBR Green. For each amplification reaction we will include a negative control to confirm the credibility of our results. This negative control will contain the same reaction mixture except the volume of DNA added is replaced by nuclease-free water.
To test for specificity, we ran RPA reactions with different RPA primer sets and target DNA. Similarly to LAMP, we will run four reactions with different combinations of primer sets and target DNA. Running triplicates of each reaction, the following RPA reactions were conducted:
- Bd RPA Primer set + Bd Gene
- Bd RPA Primer set + Bsal Gene
- Bsal RPA Primer set + Bd gene
- Bsal RPA Primer set + Bsal Gene
To test for specificity, we plan on running LAMP reactions with different LAMP primer sets and target DNA. Similarly to RPA, we will run four reactions with different combinations of LAMP primer sets and target DNA. Running triplicates of each reaction, the following LAMP reactions are conducted:
- Bd LAMP Primer set + Bd Gene
- Bd LAMP Primer set + Bsal Gene
- Bsal LAMP Primer set + Bd gene
- Bsal LAMP Primer set + Bsal Gene
For each reaction, a negative control will be included to confirm the credibility of our results. Similarly to our test for sensitivity, the negative control will contain the same reaction mixture except the volume of DNA added is replaced by nuclease-free water. The results will be visualized through gel electrophoresis. Ideally, we hypothesize that our primers in each amplification reaction will be only specific to its respective gene that it amplifies.
Detection: Cas13a vs. Cas12a
In order to increase the specificity of our detection system, we will couple our amplification reaction with a CRISPR/Cas reaction. The CRISPR/Cas system, a prokaryotic immune system, can be utilized for DNA detection. The CRISPR enzyme is extremely accurate that can effectively discriminate between targets off by down to one base pair. Ultimately, by utilizing the CRISPR/Cas system, we will be significantly increasing the specificity of our detection system. Cas12a and Cas13a are two potential CRISPR methods.
The Cas12a protein is a DNA targeting enzyme that programmatically binds and cleaves DNA. The guide RNA is a complementary sequence to the target DNA. The guide RNA helps the Cas12a enzyme to recognize the target sequence and activates the Cas12a enzyme. The CRISPR/Cas12a system recognizes a T-rich protospacer-adjacent motif (PAM) that catalyzes the maturation of its guide RNA and dsDNA break with staggered 5’ and 3’ ends. The activated Cas12a enzyme unleashes cis (for bound DNA) and trans (for other ssDNA in the solution) cleavage activity. This non-specific trans-cleavage activity can cleave a quenched fluorescence reporter (DNA sequence) in our detection medium to indicate the presence of the targeted nucleic acid sequence. Coupling CRISPR/Cas12a with RPA gives rise to a novel detection technique called DNA endonuclease-targeted CRISPR trans reporter (DETECTR) that can achieve attomolar sensitivity. The DETECTR method specifically uses LbCas12a as its CRISPR enzyme, which comes from Lachnospiraceae bacterium. (Janice S. Chen et al., 2018)
Reference: Photo Link
The Cas13a protein is a RNA targeting enzyme that programmatically binds and cleaves RNA. A guide RNA is a nucleotide sequence that is complementary to the targeted area in the RNA strand. With the help of this guide RNA, Cas13a enzyme recognizes the RNA target in solution and initiates promiscuous and rapid cleavage. The activated Cas13a enzyme unleashes indiscriminate RNA trans-cleavage, including a quenched fluorescence reporter (RNA sequence) that gives off a signal, indicating the presence of the targeted nucleic acid sequence in the sample. Coupling CRISPR/Cas13a with RPA gives rise to a novel detection technique called specific high-sensitivity enzymatic reporter unblocking (SHERLOCK) that can achieve attomolar sensitivity. It is important to note, however, that since Cas13a can only detect RNA, SHERLOCK also includes an intermediate T7 Transcription step. This step will convert the amplified DNA from RPA into RNA for which Cas13a will be able to detect. (Kellner, 2019)
CRISPR/Cas12a vs. CRISPR/Cas13a
For this year's iGEM competition, the team decided to experiment solely on Cas12a. We were given a tight period of time to perform our experiments since we were unable to perform wet-lab experiments until early October due to the current pandemic. With that in mind, our plan is to ensure that our detection system will work with CRISPR/Cas12a, and when we begin the second phase of our project for next year's jamboree, then that we test for the detection efficiency between CRISPR/Cas12a and CRISPR/Cas13a.
Reporting Mechanism: Fluorescence vs. LFA
The reporting mechanism of our detection system will rely on the CRISPR enzyme's ability to activate non-specific trans-cleavage activity upon detection of the target gene. We will incorporate a nucleic acid quenched reporter molecule in our detection system such that upon detection, our CRISPR enzyme will indiscriminately cleave the reporter molecule and produce a readout that is detectable through the naked eye. In our research, we narrowed down our reporting mechanism to two different techniques: a quenched fluorescence reporter molecule, and a lateral flow immunoassay with a FAM biotin reporter.
Detection system workflow: from amplification to detection to fluorescence signaling.
Reference: Li et al. 2019 Depending on the CRISPR enzyme being used - A CRISPR/Cas12a system would require a single-stranded DNA (ssDNA) reporter molecule while a CRISPR/Cas13a system would require a single-stranded RNA (ssRNA) reporter molecule - Our CRISPR/Cas system will trigger trans cleavage of the reporter molecule that will give out a fluorescent signal that can be detected by a fluorescence reader.
Lateral Flow Immunoassay is used for the detection of the presence of a target analyte in a liquid substance. For detection of DNA amplicons as the analyte, detection is is achieved by using a FAM-biotin labelled single-stranded nucleic acid reporter molecule.
The LFA strip consists of two lines- a control line and a test line. A positive result and a negative result is indicated by Figure 1 below.
Positive and Negative Test result assessment
LFA Mechanism - Due to the recognition and presence of The target analyte, the sgRNA/Cas- complex unleashes collateral activity. This activates the Cas complex therefore, the Cas protein cleaves the dual labelled FAM or FITC/Biotin reporter. The complexed DNA analyte, labeled with FITC/FAM and biotin, binds first to the gold-labeled FITC/FAM-specific antibodies in the sample application area of the dipstick. The gold complexes travel through the membrane, driven by capillary forces. Only the analyte captured gold particles will bind when they pass the line with the immobilized biotin-ligand molecules and generate a red-blue band over the time. Unbound gold particles migrate over the control band and will be captured by species-specific antibodies. With prolonged incubation time, the formation of an intensely colored control band appears. The mechanism of the LFA strip illustrated by the figure below.
LFA Mechanism Taken from: here
Schematic overview of options we plan on testing to create the most powerful detection platform for our point-of-care device.