Part:BBa_K4409016

gRNA (AAG+ GAPDH as guide)

This is a gRNA used in a CRISPR-Cas12 system. It mainly comprises of two components, a Cas12 handle (BBa_K2961003) which helps the gRNA to combine with a Cas12 protein, and a guide sequence (BBA_K4409017) targeting a dsDNA sequence of GAPDH. By binding with the GAPDH dsDNA (BBa_K4409006), the Cas12 enzyme is activated and DNA in the system is cleaved.

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 design a plasmid based on pET-28a plasmid by t-PCR method. We inserted the targeted sequence between the restriction enzyme site TCTAGA and CTCGAG. The PCR results show the successful plasmids construction since the site of the band is between 5000bp and 8000bp, shown in Graph 1. And the length of plasmid we design is also inside this region, exactly 5413bp. Moreover, we confirmed the sequence by gene sequencing, shown in Graph 2. All of the base pairs in the main region turns out to be accurate.

Graph 1 Confirmation of plasmid construction by DNA electrophoresis (The successful results are marked with black rectangular box)

Graph 2 Verification of BBa_K4409016 by gene sequencing. The dark blue region shows the region that is uniform between parts we design, and the region between TCTAGA and CTCGAG are the specific base we want--being marked out by black box. (The 1st role shows successful outcome, while the second role delivers erroneous outcome—inserted a gene piece)

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 3 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_K4409016 is in the third 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)