Overview of Cas9

Cas9 is one of the proteins involved in the adaptive immune system (CRISPR system) of some bacteria and most archaea. In simple terms, the Cas9 functions by binding to a small hairpin RNA called a guide RNA. The Cas9-gRNA complex then scans the DNA for a sequence complementary to the 5' amino acids of the complexed guide RNA. Once a complementary region has been found, the Cas9 cuts the DNA at that location. The Cas9 we use has been mutated such that it still complexes with the guide RNA and targets the sequence complementary to the guide RNA, but the Cas9's nuclease activity has been eliminated. Once the Cas9 finds its target site, it simply stays put.

Cas9-VP16 Characterization

Here, we describe the creation and testing of a Cas9-VP16 fusion protein. Current transcriptional level activators/repressors function by binding DNA at certain conserved sequences and influencing the transcriptional abilities of some nearby promoter. The issue with these current activators/repressors is that each one binds one unique DNA sequence. The GAL protein binds UAS sites, TetR binds TetO sites, LacI binds LacO sites, and so on. By using Cas9 as the DNA binding portion of a trans-activator, we can eliminate the need for specific binding sites by taking advantage of Cas9's unique ability to target most any DNA sequence as determined by its complexed guide RNA. The technology could be used to either target and activate endogenous sequences by creating a guide RNA which targets a sequence upstream of the promoter in question, or one could create many different guide RNAs which target different inducible promoters and activate multiple genes with one single trans-activator. Our project looks into creating a Cas9-VP16 protein and testing its ability to activate a minimal CMV promoter by targeting the fusion to two upstream binding sites using a complementary guide RNA. [[Media:Constitutive_Circuit_Diagram.png]]