Team:Freiburg/Project/crrna

CONTENT

Intoduction

One of the biggest advantages of the CRISPR-Cas system compared to other transcription activators (e.g. Zn fingers, TALEs) is that only one protein is required for targeting several DNA sites: For a new target there has to be just another crRNA. We designed a RNA plasmid containing the tracrRNA, where the crRNA can be introduced easily by digesting with BbsI and inserting two previous annealed oligos. Two of these RNA plasmids (with different crRNAs) can be fused using the iGEM biobrick system. This way it is possible to get two or more crRNA on one plasmid.
With this RNA plasmid and another plasmid containing the Cas9-effector fusion protein it is possible to target several DNA site at once by transfecting only two plasmids. This could mean the simultaneous regulation of different genes or a stricter controlling of one gen by bringing more effector domains to this gene.

Results

multiple RNA plasmid

Activation of different genes

In order to test the simultaneously activation of several genes we assembled 3 plasmids containing different fluorescent proteins. Every protein is fused to a different signal for intracellular localization. Thus, we were able to distinguish better between the different fluorescent proteins, because there will be no interference of the emitted light.

Stricter gen regulation by targeting different loci simultaneously

Summary