Part:BBa_K2644204
eGFP1T1
This is the DNA template for the sgRNA of the GFP plasmid-specific sequence. Once you want to use crispr-cas9 to reduce the GFP plasmid, you can use this DNA template to transcribe sgRNA.
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]
Usage
BBa_K2644204 carries a DNA template to produce single guide RNA (sgRNA) targeting eGFP sequence. This template was used to produce active sgRNA by in vitro T7 RNA transcription. The sgRNA was proven to be bounded to Cas9 protein and guided it to the target eGFP and then the sgRNA/Cas9 complex achieved cleavage.
Biology
It is well-known that CRISPR(Clustered regularly interspaced short palindromic repeats) is the third generation genome editing tool which is famous for specific editing ability. It works to edit or label target genes by nucleic acid-guide nuclease. There exists different kinds of nuclease together with nucleic acid in CRISPR system. So far, sgRNA/ Cas9 complex is widely applied. When identifying a target gene, sgRNA will firstly find PAM(NGG) sequence on target gene. Next, sgRNA will complement to the target sequence and then Cas9 protein cleaves the fragment. While designing sgRNA, we learn to know that sgRNA has highly conserved sequence in evolution. It is composed of two parts. One is complementary bases of target genes. This part is called spacer, which usually contains 20-25 bases. And one can choose various spacers according to the target sequences. The other part is 80nt conserved bases. Moreover, in order to make the assembly of sgRNA easier, we add T7 promotor in front of the part, which allows the in vitro transcription of sgRNA.
Results
Firstly, we designed primers based on prefix and suffix in order to construct the template of sgRNA using PCR.
Figure 1. Assembly of template of sgRNA using PCR. Lane M, marker. Lane 1, template of sgRNA. Tm=67.2℃.
Then we realized in vitro transcription of sgRNA using T7 promotor.
Figure 2. In vitro transcription of sgRNA. Lane M, marker. Lane 1, 100ng sgRNA. Lane 2, 200ng sgRNA.
After our constant effort in improving the cleavage condition, the sgRNA/Cas9 complex was proved to has quite good cleavage efficiency.
Figure 3. In vitro digestion of DNA with sgRNA/Cas9. Lane M, marker. Lane 1, eGFP plasmid. Lane 2, sgRNA:Cas9:DNA=10:20:1.
So we proceeded to make use of BODIPY, our vector, to construct our complex BODIPY/RNP and achieved high cleavage efficiency.
Figure 4. In vitro digestion of DNA with BODIPY/RNP. (A) Lane 1, eGFP plasmid. Lane 2-7 are set according to table B. Lane M, marker. (B) Experiment design.
Finally we successfully proved that the BODIPY/RNP complex had better gene editing efficiency than RNP delivered by liposome.
Figure 5. EGFP gene disruption of COS7-GFP cell line.
| None |