The CRISPR/Cas-9 system is an RNA-based DNA targeting system that is used to localize effector proteins to manipulate specific regions of DNA. For instance,null-nuclease Cas9 (dCas9) proteins can be fused to epigenome modifiers to activate or repress target genes.Cas9 proteins can locate specific target genes with the help of guide RNAs(gRNAs). Cas9 proteins are derived from different bacterial species. The most commonly used Cas9 proteins are S. pyogenes and S. aureus Cas9. However, existing Cas9 proteins have limitations such as the potential for pre-existing immunity in the human population. The lab has identified 4 novel Cas9 proteins from different bacterial species. This study aims to determine if these novel Cas9 proteins can activate the HBG1 gene within human embryonic kidney (HEK) cells given a specific gRNA. We hypothesize that in conjunction with the appropriate gRNA that targets the promoter of HBG1, these dCas9-p300 fusions can activate HGB1 expression. We first deactivated the catalytic sites in each Cas9 protein to ablate its nuclease activity and we fused it with p300, a protein that acts as an activator. Then, we transfected HEK with these dCas9-p300 plasmids and the corresponding gRNA plasmids. By running quantitative PCR tests at the end of the process, we can see which samples had higher HGB1 RNA expression.This would mean that that specific dCas9-p300 worked together with a specific gRNA sequence to activate the HBG1 gene. Overall, the discovery of new, working Cas9 proteins could help problems of immunogenicity in gene therapy patients.
