Team:WPI Worcester/Part Collection

CRISPR/dCas9 Editing System and Reporter Parts

The team created a total of 16 new BioBrick parts for submission to the registry. Each of these parts were either a part of the editing system itself or were used to test the editing system. The core of the editing system made up several parts in the collection. Each contained the dCas9/editing enzyme fusion. The three editing enzymes used were ADAR1, ADAR2, and APOBEC1. Each dCas9/editing enzyme fusion had three different variants. Each variant had different lengths of spacer sequence called XTEN Linkers: XTEN1,XTEN2, and XTEN3. The other parts of the editing system included the guide RNA, which was on the same plasmid as the gene for the RFP protein mCherry. Finally, within this collection is the different reporter plasmids used to test the editing systems. For the APOBEC1 enzyme, an eGFP molecule with a 5' untranslated region and an ACG start codon were created. Additionally, a "wild type" eGFP molecule was created with it that contained a 5' untranslated region and an ATG start codon. For the ADAR1 enzyme, an eGFP molecule with a fused globin sequence was created. This globin sequence contained an intron that when spliced out created an exon junction. It is at this junction that the different types varied. The mutant version contained a UAG codon that acts as a premature stop codon. The "Wild type" lacked the stop codon.

Above, you can see the plasmid design for the GFP reporters. They can be found here, Wild Type GFP,Mutated Start Codon GFP,Premature Termination Codon Globin with GFP, Wild Type Globin with GFP.

Above, you can see the plasmid design for our editors. They can be found here, APOBEC 1XTEN,APOBEC 2XTEN,APOBEC 3XTEN, ADAR1 1XTEN, ADAR1 2XTEN,ADAR1 3XTEN, ADAR2 1XTEN, ADAR2 2XTEN, ADAR2 3XTEN.

Above, you can see the plasmid design for the guide RNAs. They can be found here, Globin Target gRNA,GFP gRNA,Scrambled gRNA.