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| − | <p style="font-family: "Trebuchet MS", Helvetica, sans-serif;">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.</p> | + | <p style="font-family: "Trebuchet MS", Helvetica, sans-serif;">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.</p> |
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| − | <p>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.</p> | + | <p>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.</p> |
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| − | <p>We identified several different places to split SaCas9 using our model, shown below:</p> | + | <p>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.</p> |
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| − | <p>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).</p> | + | <p>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.</p> |
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| − | <p>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.</p>
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Motivation
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Design
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Results
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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.
