Difference between revisions of "Part:BBa K3017061"

Revision as of 07:33, 18 October 2019

CRISPRi sgRNA for gfp DNA binding - transcription template with terminator

This part is a transcription template CRISPRi sgRNA for gfp DNA binding with a terminator (BBa_B1002). In our project, the terminator terminates the in vitro transcription of single-guide RNA (BBa_K3017001 and BBa_K3017002) without forming secondary structures with the RNA itself, avoiding functional interferences. Users can add their own choice of promoter before this part for in vitro transcription.

dCas9 protein is directed to the specific DNA locus by a single-guide RNA (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.

Secondary structure of the transcription product of this part, predicted by NUPACK.

Complex formation between sgRNA and target DNA is depicted

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.

Handle - tracrRNA

tracrRNA is an RNA loop that acts as a handle for dCas9 to hold onto. So that the dCas9 protein is delivered to the target site together with the sgRNA. Experiments have proved that tracrRNA is strictly required for Cas9-mediated DNA interference both in vitro and in vivo. The tracrRNA forms a loop on the sgRNA after transcription to provide a scaffolding site for the dCas9 to form a duplex with the spacer.

Loop - artificial linker

Destroying the secondary structure of the handle in sgRNA could theoretically cause separation of the dCas9 protein from the sgRNA, together, removing the suppression effect. The study mentioned above had proved this hypothesis correct. The team then tried to design an artificial linker, which also forms a loop as a secondary structure, after the handle. After several trials and modifications, the research team discovered that extending the artificial loop, i.e. destroying the secondary structure, could further increase the derepression.

In our sgRNA designs, the loop is designed mostly following the study. However, our spacer is different from what the study uses. RNA secondary structure prediction by NUPACK shows that the probability of artificial linker forming secondary structures with the spacer is higher than with that of forming a loop with itself. Therefore, some nucleotides are changed to promote the formation of a loop within the artificial linker itself. Therefore, some nucleotides are changed to promote the formation of a loop within the artificial linker itself.

A corresponding antisense RNA (asRNA), K3017003, is responsible for reversing the repression effect on gfp induced by this sgRNA. Part of the antisense is complementary with the artificial linker of this sgRNA.

Sequence and Features