Due to biosafety guidelines, our wet lab experiments could not include anything that involved HPV positive human samples. We could also not work with the actual virus or the capsid protein as it requires a Biosafety Level 3 Laboratory.

Practically, we could only carry out the experiments relevant to the later steps of our kit overflow including the CRISPR/Cas12a system and the result readout on a Lateral Flow Assay strip. Following is the summary of the techniques used and experiments performed. The detailed protocol is discussed in the Protocols section under the Wetlab tab.

CRISPR/Cas12a reactions are simple enzymatic reactions that were carried out inside a Laminar Flow Hood. Prior to the conduct of these experiments, the following stock solutions of all of our reagents were made:

• E7 Target Gene
  

  E7 is the oncogene whose conservation is extremely critical for cancer progression from high risk HPV strains. As a result of our literature review and our bioinformatic analysis, we can surely say that 98 amino acids of E7 are highly conserved and can serve as an excellent biomarker. This was obtained as a gBlocks gene fragment from IDT in the form of a lyophilized powder. 
  

• ssDNA Fluorescent DNA Reporter
  

  5’- FAM - TTATT - Biotin - 3’ is the single stranded reporter whose stock solution was prepared. This is intended to be cut as a result of the trans-cleavage property of Cas12 and give us a positive HPV result in the form of an additional line on the Lateral Flow strip.
  

• Guide RNAs
  

  Guide RNAs guide the Cas enzyme to the target location where it is supposed to cleave the DNA. Three possible guide RNA sequences were figured out according to the positions of the PAM sequences found on the 5’ to 3’ strand. 
  

PCR or Polymerase Chain Reaction uses a DNA polymerase to synthesize DNA complementary to the template provided by the user. PCR mostly relies on a thermo-stable DNA Polymerase called Taq Polymerase. DNA primers are specifically designed according to the target region that needs to be amplified. These primers provide a starting point for DNA synthesis. The two primers - forward and reverse, bind to opposite strands of the template DNA on the basis of complementarity. Taq polymerase is then responsible for the extension of the DNA from the primers.

Steps involved in PCR

PCR involves three basic steps:

• Denaturation - The two DNA strands are separated by high temperature (96C). This provides single strand templates for the primers to bind to.
  
• Annealing - Cool the system down to 55-60 C to allow the primers to bind on the basis of complementarity.
  
• Extension - Raise the reaction temperature to 72C for the Taq polymerase to work and extend the primers thereby synthesizing new DNA with the help of dNTPS present.
  

This cycle is repeated 25-35 times. After each cycle, the new DNA serves as the template. This doubles the DNA in each cycle.

DNA elution is the process of extraction of DNA (in this case - after gel electrophoresis) by washing with a solvent. 
After the DNA is eluted, its concentration is figured out using a NanoDrop. A NanoDrop helps to quantify DNA on the basis of the amount of 260 nm light absorbed by the sample. For a dsDNA sample, a sample with optical density = 1 equates to 50 ng/uL of DNA. New NanoDrops require only 2uL of the sample solution for the quantification. Elution buffer is used as the blank in all measurements using a NanoDrop.

Gel electrophoresis is a laboratory technique that is used to separate a mixture of DNA on the basis of their molecular size. The separation is done by pushing the molecules through a gel by using an electric field. The molecules travel through with speed that is inversely proportional to their size. Nucleic acids are negatively charged and are pushed towards the positive terminal of the setup. DNA samples are loaded into wells created in the gel (Agarose in this case). The gel is stained with a DNA binding dye because of which the loaded gel can be seen as bands in the gel. One well in the gel is reserved for the DNA ladder which contains DNA of known lengths as a standard reference. The shorter pieces of DNA travel faster and reach the bottom faster. After the separation, the gel is placed under UV light where the DNA bands are clearly visualized because of their glow.The gel can also be visualized in a gel doc. A gel doc or Gel documentation, or gel imaging, systems are used to record and measure labeled nucleic acid and protein in various types of media such as agarose, acrylamide or celluloseIn our system, gel electrophoresis was used to visualize a band at the 250 base level on the ladder to ensure that the target DNA was amplified after PCR and that no error occured. Gel electrophoresis was also used to ensure that our target DNA was cut after the CRISPR reaction. After adequate incubation of the CRISPR reaction solution, the solution was loaded and no band was obtained.

CRISPR or Clustered Regularly Interspaced Short Palindromic Repeats is a genome editing technique that has widely come into use. This system has been adapted from a naturally existing bacterial immune defense system. CRISPR integrates the use of a guide RNA and a nuclease called Cas which cuts the target DNA wherever the guide RNA binds.We have utilized the CRISPR/Cas12a system for the diagnosis of HPV16. The Cas12 system has been utilised due to its special trans-cleavage property due to which any ssDNA present in the solution is also cleaved. CRISPR reactions in the lab mostly involve the formation of a RiboNucleic Protein or RNP complex in the first step. For this, the Cas enzyme and the guide RNA are allowed to associate with each other. After the RNP complex is made, the target DNA and the ssDNA reporter are added which are then cleaved by the Cas enzyme.

The results of the CRISPR reaction are visualized on a Lateral Flow Assay based dipstick ordered from Milenia Biotec. Capillary forces drive the sample from the sample pad to the conjugate pad. The conjugate pad consists of Anti-FITC antibodies bound to gold nanoparticles. The 5’- FAM end of our reporter selectively binds with the Anti-FITC antibodies. As the sample moves further, it encounters the test line which is lined with streptavidin that interacts with the 3’ -Biotin end of our reporter.When E7 is present in the reaction system i.e. the sample is positive for HPV, some of the reporter would be cleaved because of the trans cleavage property of Cas12. The cleaved reporter would give an indication on the control line because of Anti-FITC antibodies interacting with IgG antibodies. The remaining uncleaved reporter would give an indication on the test line because of Biotin-Streptavidin binding. Thus, two lines would be observed.When E7 is not present i.e. the sample is negative for HPV, our solution would only contain uncleaved reporters. The 3’- Biotin end of these reporters would interact with Streptavidin and give a visual signal only on the test line.