Design
We applied our split protein pipeline [link to this] to determine promising split locations for saCas9. We chose 10 candidate integrase split sites and 4 candidate RDF split sites, and cloned these into our mammalian expression plasmid backbones that included fusion to our sets of dimerizable domains. Here, we focused on creating FKBP-FRB conditionally dimerizable saCas9 variants, as these domains are small enough to still fit into the AAV limit. We independently tested the functionality of these conditionally dimerizable proteins using a fluorescent reporter plasmid.
We planned to test our split saCas9 using a traffic light reporter developed by the Scharenburg lab1. It was originally designed for assessment of activity of a different endonuclease (spCas9), which has a different PAM sequence compared to saCas9. Thus, we designed a sgRNA that would recognize a complementary sequence in the traffic light reporter corresponding to the saCas9 PAM criteria, and would recruit the saCas9 protein to produce a DSB. This DSB could be repaired either through non-homologous end joining (NHEJ) or homology directed repair (HDR) pathways.
For testing the functionality of our conditionally dimerizable saCas9, we used the traffic light reporter containing fluorescent proteins - EGFP and mCherry. Initially, with an inactive saCas9, neither EGFP nor mCherry would be expressed, as there is a premature stop codon within the EGFP sequence. Presence of the inducer would lead to activity of saCas9, such that it would create a DSB.
If the DSB was repaired by NHEJ, a two base-pair frameshift would occur; the EGFP would be rendered out of frame and would be expressed as a “gibberish” sequence, while the mCherry would be in frame and would be expressed. If the DNA was repaired by HDR, the DSB could be repaired by copying off a correct sequence using a separate EGFP donor template; the EGFP would thus be expressed, while the mCherry would be out of frame and not expressed. This way, not only we could assay for a functional, conditionally dimerizable saCas9, but also to assay its ability to carry out NHEJ and HDR.
