1  Project Goal and the product

Our project aims to combine a CRISPR-Cas12a system with a cell-free system to identify two circRNA biomarkers of breast cancer, hsa_circ_0001785 and hsa_circ_0001982. This detection kit is designed to be sensitive for minimally invasive samples like blood. It is sensitive, cheap, and widely applicable, and can be used by ordinary people at home. Our kit can be used to screen breast cancer for the general public, especially those with high risk factors. Detection of hsa_circ_0001982 is also useful for triple-negative breast cancer (TNBC) patients to check whether they can benefit from conventional chemotherapy.  

Our final product consists test tubes, reagents and a detection device. The test kit provides all cell-free ingredients needed for the CRISPR-Cas12a system to work. A sample such as blood can be added into the tube with other reagents, negative controls and positive controls are provided by our product to ensure the reliability.  If the target circRNA such as hsa_circ_0001982 is present, a fluorescence signal will emit. The detection device is designed for users to observe fluorescent signals in a convenient and safe way.

The CRSPR-Cas12a system in the tube contains a specially-designed igRNA, Cas12a enzyme, dsDNA substrate designed in accordance with the igRNA, and a reporter DNA (fluorescence probe). The igRNA can sensitively and specifically bind to the target circRNA (trigger RNA) and then release a portion of its sequence to bind with the dsDNA. This binding activates Cas12a enzyme to cut the probe, resulting in a fluorescence signal (Figure 1).

 

 

Figure 1 Schematic representation of the CRISPR-Cas12a system in our project

 

In our project, we eventually developed 5 detection systems for detecting either hsa_circ_0001982 or hsa_circ_0001785 using different dsDNAs. The 5 systems are classified into two types, as shown below.

-Type 1: System utilizing synthetic dsDNA

(1)  System (1982 trigger GAPDH): trigger RNA is hsa_circ_0001982, and dsDNA is GAPDH (abbreviated as: 1982 trigger GAPDH).

(2)  System (1785 trigger GAPDH).

-Type 2: System utilizing non-synthetic dsDNA

(1)  System (1982 trigger 1785).

(2)  System (1785 trigger 1982).

(3)  System (1982 trigger GAPDH).

 

2 Summary of lab proof

In order to find out the optimal venue which our detection kit can achieve its desired effects, we tested the sensitivity of these systems with cell extracts and supernatants of the breast cancer cell line mcf7 and the TNBC cell line MBA-MD-231. Cell extracts are comparable to tissue samples in real world, while cell supernatants can simulate blood and urine samples of the patients. 

Our lab results showed that the sensitivity was high enough for all five systems to detect target circRNAs in cell extracts and supernatants from breast cancer cells. Systems targeting hsa_circ_0001982 were more sensitive than those targeting hsa_circ_0001785 when detecting low-concentration cell extract samples and cell supernatants. Systems with non-synthetic dsDNA were more sensitive to cell supernatants than those with synthetic dsDNA.

 

3 Sample preparation for experiments

The TNBC cell line MBA-MD-231 and the breast cancer cell line mcf7 were gained from Shanghai Cell Bank. Cell extracts were obtained by extracting the total RNA (500ng/µl) from MBA-MD-231 cells through the guanidinium-acid-phenol extraction method. The cell supernatant was obtained by taking the culture medium of the cancer cells.

The non-synthetic dsDNAs, hsc_circ_0001785 dsDNA, hsc_circ_0001982 dsDNA and GAPDH dsDNA, utilized in this experiment were obtained through reverse transcription and isothermal amplification of the RNA extracted from MBA-MD-231 and mcf7 cells. The synthetic dsDNA, GAPDH dsDNA, was obtained through annealing of synthetic forward and reverse primers for GAPDH.

To get the igRNAs, we expressed their corresponding recombinant plasmids through the cell-free TXTL method. The plasmids were constructed through inserting igRNA sequences between Xba1 and Xbo1 enzyme cutting sites into the vector plasmid pET28a+. The plasmids were as follows:

1.  plasmid for igRNA which had a guide sequence of GAPDH and an inactive part for activation by hsa_circ_0001982.

2.  Plasmid for igRNA which had a guide sequence of GAPDH and an inactive part for activation by hsa_circ_0001785.

3.  Plasmid for igRNA which had a guide sequence of hsa_circ_0001982 and an inactive part for activation by hsa_circ_0001785.

4.  Plasmid for igRNA which had a guide sequence of hsa_circ_0001785 and an inactive part for activation by hsa_circ_0001982.

For all experiments, the positive control was procured by adding a circRNA mimic instead of cell extract or cell supernatant into the system as trigger RNA. The negative control was being set up by adding nuclease-free water instead of trigger RNA into the system.

 

4 Experiments

4.1 Test of systems utilizing synthetic dsDNA

The two systems both utilized the synthetic GAPDH dsDNA, and each with igRNA (1982 trigger GAPDH), or igRNA(1785 trigger GAPDH). In order to test the least concentration of the trigger RNA needed to activate the two systems, we used 4 concentration gradients of trigger RNA (cell extract 1, 0.5, 0.05, and 0.125µl). Cell supernatants were also used to test the sensitivity of the systems. The results are shown below in Figure 2.  

 

Figure 2 Results of igRNAs of 1982 trigger GAPDH, and 1785 trigger GAPDH (trigger RNAs are non-synthetic)

Trigger RNAs from left to right: 1) cell extract 1µl 2) cell extract 0.5 µl 3) cell extract 0.05µl 4) cell extract 0.125µl 5) cell supernatant (MBA-MD-231) 6) cell supernatant (mcf7) 7) positive control 8) negative control

The results showed that hsc_circ_0001982 was more efficient as a trigger RNA than hsc_circ_0001785 since the fluorescence in the first set of tubes was much stronger than that in the second set of tubes. Trigger hsc_circ_0001982 could be detected by the system even given an extremely low concentration of trigger RNA (0.125µl cell extract). However, trigger hsc_circ_0001785 could be detected only when at least 0.5µl of the cell extract was added. Trigger hsc_circ_0001982 could be detected by the system in cell supernatants as well, while the fluorescence signal of trigger hsc_circ_0001785 was low for the supernatants.

 

4.2 Test of systems utilizing non-synthetic dsDNA

Non-synthetic dsDNA of hsc_circ_0001982, hsc_circ_0001785 and GAPDH sequence were used for these systems. We created three models of igRNAs to prove our concept: 1982 trigger 1785, 1785 trigger 1982, and 1982 trigger GAPDH. The results are shown below in Figure 3-5.

 

Figure 3 Results of igRNAs of 1785 as dsDNA and 1982 as trigger RNA

Trigger RNAs from left to right: 1) cell extract 1µl 2)cell extract 0.5µl 3)cell supernatant(mcf7) 4)cell supernatant (MBA-MD-231) 5)positive control 6)wastage; dsDNA utilizes isothermally amplified product (with 1785 primer)

 

 

Figure 4 Results of igRNAs of 1982 as dsDNA and 1785 as trigger RNA

Trigger RNAs from left to right: 1) cell extract 1µl 2)cell extract 0.5µl 3)cell supernatant(mcf7) 4)cell supernatant (MBA-MD -231) 5)positive control 6)wastage; dsDNA utilizes isothermally amplified product (with 1982 primer)

 

 

Figure 5 Results of igRNAs of GAPDH as dsDNA and 1982 as trigger RNA

Trigger RNAs from left to right: 1) cell extract 1µl 2)cell extract 0.5µl 3)cell supernatant(mcf7) 4)cell supernatant (MBA-MD -231) 5)positive control 6)wastage; dsDNA utilizes isothermally amplified product (with GAPDH primer)

 

All of them showed fluorescent signal, including in cell extracts and supernatants of cancer cells MBA-MD-231 and mcf7, which indicated all systems were able to be applied to real-life usage. However, through comparison of the intensity of these signals, we found out that hsc_circ_0001982 acts as a better trigger than hsc_circ_0001785; the supernatant of mcf7 cells was more efficient than the cell supernatant of MBA-MD-231.