Part:BBa_K4409009

igRNA (AAG+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

We constructed a plasmid based on pET-28a plasmid by inserting the sequence of this part between the restriction enzyme sites using t-PCR method. PCR results showed the plasmid was constructed successfully since the site of the band is between 5000bp and 8000bp, shown in Graph 2, and the length of plasmid we constructed was also in this region, exactly 5455bp. Moreover, we confirmed the sequence by gene sequencing, shown in Graph 3. All of the base pairs in the main region turns out to be accurate.

Graph 2 Confirmation of plasmid construction by DNA electrophoresis (The black rectangle shows the successful plasmid construction result)

Graph 3 Verification of the BBa_K4409009 by gene sequencing method (The dark blue region shows the region that is right, and the region between TCTAGA and CTCGAG are the specific bases we want)

A CRISPR-Cas12 system using this igRNA produced fluorescence significantly bigger than negative control groups. The fluorescence produced is shown in graph 4 and table 1-2.

Graph 4 The final fluorescence level of each test tubes (The first test tube is the positive control group, the last test tube is the negative control group, and BBa_K4409009 is in the second test tube)

Table 1 Relative Brightness of fluorescence produced (Error bars are printed based on standard deviation)

Table 2 Percentage difference of fluorescence [Calculated by the following formula: (Brightness-negative control brightness)/negative control brightness] (Error bars are printed based on standard deviation)