Part:BBa_K2019000
Histidine Tagged WT Staphylococcus Aureus Cas9 Protein
Part BBa_K2019000 consists of a histidine tagged wild-type staphylococcus aureus CRISPR associated protein 9 (saCas9) codon optimized for escherichia coli.
Usage and Biology
Usage and Biology
CRISPR-Cas9 has one of the most promising tools for genome editing in the field of synthetic biology due its ability to cut DNA with extremely low off target effects and high efficiency. The most commonly used CRISPR endonuclease is Cas9 from the bacteria streptococcus pyogenes (spCas9). However, one major limitation of saCas9 is its size. The gene that encodes for WT spCas9 is roughly 4.2 kb and produces a protein which is roughly 155 kDa, which runs into issues for several delivery mechanisms, including adeno-associated viruses (AAV) and outer membrane vesicles (our own delivery mechanism). saCas9 helps alleviate the issue of size of Cas9 with retaining the same functionality as spCas9. saCas9's gene is only 3.2kb, andd the protein size is around 113 kDa, over 40kDa less than spCas9. Furthermore, the PAM sequence for saCas9 is NNGRRT, compared to the NGG PAM sequence for spCas9. Statistically, the increased complexity of the PAM sequence of saCas9 will lead to less off-target effects compared to its counterpart spCas9.
We have confirmed that this sequence encodes for the correctly sized saCas9 protein along with demonstrating its functionality through a functionality test using parts BB_K2019001 and BB_K2019002. Figure X includes a western blot of his-tagged proteins in 2 different E. Coli cell lines, Top10 and BL21, that express our saCas9 in a tet (BBa_R0040) and pET28 backbone, respectively. As one can see, there are correctly sized bands around 110 kDa, confirming that our protein is correctly sized. Moreover, we ran a functional test of our saCas9 expression system in the tet backbone. In this experiment, we had BB_K2019001 and BB_K2019000 cotransformed into E. Coli along with BB_K2019002 and BB_K2019000 cotransformed into a different E. Coli cell line. If our saCas9 is functional, the cell line with BB_K2019002 and BB_K2019000 should have less fluorescence than BB_K2019001 and BB_K2019000. As one can see, the relative fluorescence is significantly less for the cell line with BB_K2019002 and BB_K2019000 (labeled "guide in the graph below) , suggesting that our saCas9 successfully cut the mRFP protein in BB_K2019002.
Figure 1: Relative fluorescence of BB_K2019000+BB_K2019001 ("template"), BB_K2019000+BB_K2019002("guide"), and BB_K2019000 ("Cas9 only")
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 627
Illegal BglII site found at 780
Illegal BglII site found at 926 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 200
Illegal AgeI site found at 2756 - 1000COMPATIBLE WITH RFC[1000]
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