Difference between revisions of "Part:BBa K3017001"
| Line 7: | Line 7: | ||
| + | <h2>Spacer - crRNA</h2> | ||
| − | + | [[File:T--Hong_Kong_HKUST--sgDNA_GFP_white.jpeg|thumb|Complex formation between sgRNA and target DNA is depicted]] | |
| − | + | ||
| − | + | ||
| − | + | ||
| − | + | ||
<p>crisprRNA(crRNA) is also commonly referred to as the spacer. When choosing the target binding region, we considered mainly 2 factors, namely the location of the PAM sequence and the suppression effect upon binding.</p> | <p>crisprRNA(crRNA) is also commonly referred to as the spacer. When choosing the target binding region, we considered mainly 2 factors, namely the location of the PAM sequence and the suppression effect upon binding.</p> | ||
<p>The research shows CRISPRi suppression effect is the strongest 35nt upstream start codon of the coding region. However, the area upstream of our coding region is a generic constitutive promoter. To avoid non-specific binding, we compromised the suppression efficiency and chose a region shortly after the start codon, where suppression is only a few percents weaker than the ideal region. We found a PAM sequence (TGG) 27nt into <i>gfp</i> part BBa_E0040, which lead to a sgRNA binding region spanning 20bp, 7nt into CDS. To accommodate the PAM sequence in BBa_E1010 <i>mrfp</i>, the spacer is arranged on the opposite DNA strand, 14nt into the gene. When this part, specific to <i>gfp</i> is transcripted, GFP is suppressed.</p> | <p>The research shows CRISPRi suppression effect is the strongest 35nt upstream start codon of the coding region. However, the area upstream of our coding region is a generic constitutive promoter. To avoid non-specific binding, we compromised the suppression efficiency and chose a region shortly after the start codon, where suppression is only a few percents weaker than the ideal region. We found a PAM sequence (TGG) 27nt into <i>gfp</i> part BBa_E0040, which lead to a sgRNA binding region spanning 20bp, 7nt into CDS. To accommodate the PAM sequence in BBa_E1010 <i>mrfp</i>, the spacer is arranged on the opposite DNA strand, 14nt into the gene. When this part, specific to <i>gfp</i> is transcripted, GFP is suppressed.</p> | ||
| − | |||
| − | |||
Revision as of 03:36, 15 October 2019
CRISPRi sgRNA for gfp DNA binding - transcription template
dCas9 is directed to the specific DNA locus by a sgRNA, where it binds to suppress downstream gene expression. With reference to the research on reversible CRISPRi switch, we redesigned the traditional sgRNA by adding an artificial linker behind crRNA and tracrRNA and modified the 3-component-sgRNA to suit our suppression purpose. Our design of sgRNA is compatible with spCas9.
Spacer - crRNA
crisprRNA(crRNA) is also commonly referred to as the spacer. When choosing the target binding region, we considered mainly 2 factors, namely the location of the PAM sequence and the suppression effect upon binding.
The research shows CRISPRi suppression effect is the strongest 35nt upstream start codon of the coding region. However, the area upstream of our coding region is a generic constitutive promoter. To avoid non-specific binding, we compromised the suppression efficiency and chose a region shortly after the start codon, where suppression is only a few percents weaker than the ideal region. We found a PAM sequence (TGG) 27nt into gfp part BBa_E0040, which lead to a sgRNA binding region spanning 20bp, 7nt into CDS. To accommodate the PAM sequence in BBa_E1010 mrfp, the spacer is arranged on the opposite DNA strand, 14nt into the gene. When this part, specific to gfp is transcripted, GFP is suppressed.
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]