Part:BBa_K5223009
HQT3-sgRNA
HQT3-sgRNA is designed for the knockout of NbHQT3 (NbL13g01170.1). Like the others, this sgRNA is part of the CRISPR-Cas9 system and specifically targets the HQT3 gene for gene-editing purposes.
Biology and Usage
| HQT3-sgRNA | |
| Function | The double-gene knockout plant of HQT3 |
| Use in | Nicotiana benthamiana |
| Backbone | BGFastCas9 SS Plant |
| Derived from | Artificial synthesis |
Design and Properties:
Figure 1: Schematic Diagram of Gene Circuit
A
B
C
Figure 2(A) Detection Results of HQT3 (B) Sequence Alignment Diagram (C) Electrophoresis Image
The results in Figure 2 demonstrate that tobacco plants successfully underwent gene knockout, achieving the expected
outcomes.
Experimental Approach:
We used homologous recombination for vector construction. The gene-editing vector used was the BGFastCas9 SS Plant from BIOGROUND Biotechnology.(1) Primer Design:
We pre-selected four target sequences corresponding to the genes (about 20 bp, complementary to the gRNA) and incorporated them into primers designed to amplify intermediate sequences from the vector.
Table 1 Primer Design
| Component | Volume |
| 2x High-Fidelity Enzyme | 25 μL |
| Forward Primer | 2 μL |
| Reverse Primer | 2 μL |
| Intermediate Vector osgRNA-U6 | 1 μL |
| ddH2O | Make up to 50 μL |
Table 2 PCR System
PCR cycling conditions: 98°C for 3 min; (98°C for 10 s, 50-58°C for 20 s, 72°C for 1 min) for 30 cycles; 72°C for 5
min; stored at 4°C. After PCR amplification, gel recovery was performed.
(3) Homologous Recombination:
| Component | Volume |
| 5x CE Buffer II | 2 μL |
| Exonuclease II | 1 μL |
| Linearized Vector Fragment | 150 ng |
| Single Target Fragment | 70-80 ng |
| ddH2O | Make up to 10 μL |
Table 3
Incubate at 37°C for 30 min. The recombinant solution was stored at -20°C.
(4) Transformation and Screening:
1. Thaw competent DH5α cells on ice, add 5 µL of the recombinant product to 50 µL of thawed cells, and incubate on
ice for 30 minutes.
2. Heat shock at 42°C for 60 seconds, then place on ice for 5 minutes immediately.
Add 0.8 mL of LB medium to the mixture and incubate at 37°C for 1 hour with shaking.
3. Centrifuge at 6000 rpm for 3 minutes, discard approximately 700 µL of the supernatant and resuspend the cell
pellet.
4. Spread the cell suspension evenly on LB agar plates containing kanamycin. After 30 minutes of incubation, invert
the plates and incubate overnight at 37°C.
5. After overnight incubation, pick colonies for PCR identification of positive clones.For colonies with the correct
band, send samples for sequencing. Positive clones were expanded and used for plasmid extraction.
For colonies with the correct band, send samples for sequencing. Positive clones were expanded and used for plasmid
extraction.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
| None |