The overall goal of our project is to prove that single base nucleotide changes can occur in an organism when editing enzymes are coupled with the CRISPR/dCas9 system. Designing effective plasmids and reporters was the first step in ensuring that we would be able to accurately show this proof of concept.

Reporters

We designed 2 reporters, one for each editing enzyme. Each reporter is designed to emulate certain point mutations that can occur and cause diseases. The APOBEC reporter imitates a mutation to a start codon and the ADAR reporter imitates a mutation that creates a premature termination codon (PTC). For successful targeting by the CRISPR/dCas9 complex onto their mRNA each reporter contains a 5’ untranslated region (UTR). They also both used a green fluorescent protein (GFP) as the editing identifier. When expressed in the cell without presence of the editing complexes, the reporters should not produce GFP. Thus, the cells would not fluoresce. Once the editing complex binds and edits the reporters, however, GFP should then be translated and the cells will glow green.

APOBEC Reporter

For our APOBEC experiments we used the following reporter. The reporter is a GFP sequence with the 5’ UTR for the CRISPR/dCas9 to bind to. The only change we made to the GFP sequence was mutating the start codon (ATG) to an ACG codon. The reason for this being that when the ribosome would bind to the reporter’s mRNA, translation should not occur since there is no start codon. Therefore, we should not see any fluorescence in the transfected cells.

Once the CRISPR/dCas9 and APOBEC fusion is added to the cells the CRISPR/dCas9 will be able to bind to the 5’ UTR of the reporter. APOBEC will then make its C to U edit on the ACG codon, making it an AUG codon, which is a start codon in mRNA. Now when the ribosome binds, the GFP should be translated and the cells will fluoresce.

ADAR Reporter

For our ADAR experiments we designed a slightly more complex reporter. The reporter is a beta globin sequence with a GFP sequence after as well as the 5’ UTR for CRISPR/dCas9 binding. The reason for using beta globin is because it has an intron. When this intron is spliced out during transcription it will leave an exon junction. This is important because we put a PTC (UAG codon) before the exon junction. When the ribosome starts to translate the protein it will see that there is an exon junction after a stop codon. The ribosome recognizes that this should not occur and will mark the mRNA for degradation. The mRNA will be degraded and no protein will be translated. Resulting in no fluorescent cells.

Once the CRISPR/dCas9 and ADAR fusion is added to the cells the CRISPR/dCas9 will be able to bind to the 5’ UTR of the reporter. ADAR will make its A to I edit on the PTC, changing the UAG codon to a UIG codon. I, inosine, is read as a G by the ribosome, so the new UGG sequence would produce a tryptophan instead of being a PTC. Without the PTC the ribosome should be able to translate the rest of the protein, including the GFP. With the GFP translated the cells should now fluoresce.

Plasmids

To accomplish our experiment we designed three different plasmids. The components of each were cloned in E. coli cells and then miniprepped. All three plasmids were then transfected into our chassis, HEK293T cells.

dCas9 Plasmid

This is our first plasmid. It contains one of the editing enzymes, either ADAR1, ADAR2, or APOBEC1, fused to dCas9. The enzyme and dCas9 have 1-3 repeats of an XTEN linker to allow the enzyme to edit a certain distance away from dCas9. Downstream on the same plasmid is the rtTA gene and promoter.

One of the key parts of our system is that we wanted it to be tunable. We don't want the enzymes to be editing at all time, especially if something was to go wrong. Regulation is the purpose of the rtTA gene in this plasmid. rtTA, which stands for reverse tetracycline trans activator, is constitutively expressed. It acts as a repressor binding to the tetracycline responsive element (TRE) promoter and preventing transcription of the editing enzyme and dCas9. However, when doxycycline is added to the system it will bind to the rtTA, removing it from the promoter and allowing transcription. Editing will not occur unless we introduce doxycycline into the cells.

mCherry and Guide RNA Plasmid

This plasmid contains our guide RNA and mCherry. The guide RNA is complementary to the target editing site and will help direct the dCas9 there. mCherry is type of RFP and is constitutively expressed. This is our transfection marker and shows that the plasmids have sucessfully entered the cells.

Reporter Plasmid

The third plasmid is a GFP reporter. Two of the reporters, the betaglobin wild type and GFP reporter with a normal start codon both do not require editing. The other two, the ACG GFP reporter and the PTC globin reporter should show fluorescence if editing was successful.