Part:BBa_K4409010

igRNA (CT+GAPDH as guide+ hsa_circ_0001982 as trigger)

This is an RNA-interacting guide RNA (igRNA). It is used in a CRISPR-Cas12 system. It mainly comprises the following three parts. The first part (BBa_K2965036) is the Cas12 handle which combines with Cas12 protein. The second part (BBA_K4409000) is a guide sequence targeting a DNA sequence of GAPDH. The last part (BBa_K4409004) is a control sequence made up of VR1, loop and VR2 regions in sequence. VR1 and VR2 regions can specifically bind to the RNA trigger which is a unique sequence of hsa_circ_0001982. The loop binds to a portion of the guide sequence. When hsa_circ_0001982 is present in the CRISPR-Cas12 system, the control sequence binds with it and the igRNA is activated. The loop then unblocks the guide. The guide binds to a GAPDH dsDNA with the PAM sequence, and activates the Cas12 protein, resulting in the cleavage of dsDNA and ssDNA in the system.

Graph 1 Figure Schematic representation of the inactive (A) and active(B,C) igRNA in a CRISPR-Cas12 system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Usage and Results

In order to test the efficiency of this sequence, we put this sequence into the Cas12a system we constructed. Coupled with Cas12a enzyme, probe, buffer, and dsDNA, fluorescence is being produced. We use ImageJ to measure the fluorescence level in each tests tube. From left to right, each test tube contains different concentration of circRNA from different sources, marked as following: Cell extract from cancer cell 1μl, cell extract from cancer cell 0.5μl, cell extract from cancer cell 0.25μl, cell extract from cancer cell 0.125μl, cell supernatant from cancer cell 1, cell supernatant from cancer cell 2, positive control group, negative control group. Concentration of all other substance is controlled the same.

Graph 1 Fluorescence reaction diagram)

The fluorescence produced is recorded in the following table. The value of d% is calculated as the following formula

Conclusion

Since when circRNA is present, the fluorescence produced is significantly higher than the negative control groups when circRNA is not present. This proves that igRNA we design is a workable one since only when circRNA is present, fluorescence can be produced. This serves as a switch for the whole system.