Design
We decided to focus on the CRISPR/Cas9 system of genome editing because it is easily customizable. The sgRNA recognizes a complementary sequence that is upstream of a PAM sequence (protospacer adjacent motif). The sgRNA then recruits Cas9 to make a double stranded break at the target sequence. Changing the target of the CRISPR/Cas9 system is as easy as modifying the sgRNA to recognize a sequence of interest.
The most widely studied version of Cas9 is streptococcus pyogenes cas9 (SpCas9). However, spCas9 is over 4 kb long, and its size presents limitations during viral delivery. Packaging spCas9 into the commonly used adeno-associated virus (AAV) vector presents challenges because of the AAV vector’s limited cargo size. Therefore, we decided to apply our split methodology to a recently identified variant of Cas9: staphylococcus aureus cas9 (SaCas9). It is about 1 kb shorter than SpCas9, making it easier to package into the AAV vector.
We identified several different places to split SaCas9 using our model, shown below:
We plan to test our split saCas9 using a traffic light reporter developed by the Scharenburg lab1. It was originally designed for a different endonuclease, but we designed a sgRNA that would recognize a complementary sequence in the traffic light reporter and recruit the Cas9 protein to produce a DSB. This DSB is repaired either through non-homologous end joining (NHEJ) or homology directed repair (HDR).
Originally, neither GRP nor mCherry is expressed. If the DSB is repaired by NHEJ, a two base-pair frameshift occurs. The GFP will therefore be rendered out of frame and will be regarded as gibberish and mCherry will now be in frame, and will therefore be expressed. If HDR occurs, the DSB will be repaired using a separate GFP template donor, and GFP will be expressed. An important note here is that we care more about the SaCas9 actually producing its desired activity and making the DSB, so either color being expressed will prove the success of our induced SaCas9. This traffic light reporter allows us to not only verify our split protein system, but also characterize the repair activity.
