CRISPR 3.0 plasmid

Summary

Multiple aspects of CRISPR 3.0 technology need to be taken into account and included when when designing a plasmid that will be able to perform edits within a cell. These include sequences that code for the Cas9 protein, the reverse transcriptase, and the prime editing guide RNA (pegRNA). The pegRNA itself will have to have specific sequences to allow it to ligate to DNA following nicking, as well as functioning as both a primer and template for the reverse transcriptase. More information about how to design pegRNA can be found here.

In Our Lab

We will be using addgene/dnasu to create and order our plasmids. The plasmid(s) will need to contain eGFP, the prime editing fusion protein (Cas9 H840A Nickase and M-MLV Reverse Trancriptase), a designed pegRNA, and designed secondary sgRNA used to nick the unedited strand following editing.

When it comes to designing the pegRNA and secondary sgRNA, the tool PegFinder can be used. Protocol regarding designing pegRNA specific to your desired edit using pegFiner can be found on the protocols page. Once designed on pegFinder, your desgined pegRNA can be incorporated into an Addgene plasmid listen on PegFinder's website. However, likely a separate plasmid will need to also be transfected along with withe pegRNA plasmid. The second plasmid will need to contain sequences for eGFP and the prime editing fusion protein (Cas9 H840A Nickase and M-MLV Reverse Trancriptase) - such as the pCMV-BE2 plasmid that we possess. (we may need to purchase a new updated plasmid though).

Transferred from Old Site:

Summary

Plasmids are singular, circular pieces of DNA most commonly found in prokaryotes such as bacteria. Their circular structure is different from the linearized forms of DNA found in eukaryotes, but plasmids are still able to be expressed by eukaryotic cells if the plasmids are able to be introduced into the cells' nuclei. We have the ability to design plasmids virtually (such as through _____ programs) to contain specific genes of interest to us, and we can then order these plasmids to be developed and shipped to our lab. Once we obtain the initial plasmid DNA, we can utilize our transformation and maxi prep protocols to obtain a larger stock of the plasmid. From this stock, we can use or cell culture and transfection techniques to introduce the plasmid into our cultured cells, ideally resulted with our genes of interest being expressed in the cultured cells.