Histidine-tagged Wild-Type Staphylococcus Aureus CRISPR Associated Protein 9 (saCas9)
saCas9 is a RNA guided endonuclease that has proven to be an incredibly viable genome editing tool. Compared to the more generally used streptococcus pyogenes cas9 (spCas9), saCas9 is roughly 40 kDa smaller and only sacrifices slight levels of efficiencies. The construct includes the wild type sequence of saCas9 codon optimized for Escherichia coli. Furthermore, saCas9 has a his6 tag fused to the N-terminus of the protein, which can be utilized for protein purification.
gRNA Template with Constitutively Expressed mRFP
This construct includes a mRFP gene with a constitutive promoter (BBa_J04450) upstream of the BBa_J23119-template-sgRNA scaffolding device. The template sequence is 20 bp region flanked by BsaI at the 5’ and 3’ ends. These BsaI restriction sites allow for one to insert 20 bp guide sequences in place of the template via Golden Gate Assembly. These guide sequences will then be constitutively expressed and will guide saCas9 to target specified sequences.
gRNA Guide Sequence with Constitutively Expressed mRFP
In the presence of saCas9, the constitutively expressed gRNA guide sequence will direct saCas9 to cut the mRFP protein, causing the cell line to lose its red fluorescence. Two overlapping primers were designed in order to insert a 20 bp guide sequence into the gRNA template construct (BBa_K2019001) via Golden Gate assembly. The guide sequence targets the 130-150bp region of the mRFP gene and has an on-target score of 74.9 and off-target score of 99.1 according to Benchling.
Periplasm Directed saCas9 via TorA Signaling Sequence (TorA-saCas9)
TorA is a signaling sequence that utilizes the post-translational twin arginine translocation pathway to direct a fused protein to the periplasm of the cell. In this construct, the TorA sequence is fused to the N-terminus of saCas9 in an attempt to direct saCas9 to the periplasm of Escherichia coli. The saCas9 gene is from part BBa_K02019003; thus it has a histidine tag on the N-terminus of the protein.
Periplasm Directed saCas9 via DsbA Signaling Sequence (DsbA-saCas9)
DsbA is a 57 bp signaling sequence that operates in the SRP, a cotranslational pathway that utilizes certain aspects of the sec pathway to send fused proteins to the periplasm of the cell. Similar to K2019003, DsbA is also fused onto the N-terminus of saCas9 (from part BBa_K02019003). Furthermore, expression of DsbA-saCas9 can be detected utilizing this his tag on the N-terminus of saCas9.
Periplasm Directed saCas9 via Ycdo Signaling Sequence (Ycdo-saCas9)
The signaling sequence Ycdo utilizes the cotranslational sec pathway to direct proteins to the periplasm of the cell. In this construct, Ycdo is fused to the N-terminus of saCas9. This Ycdo-saCas9 can be detected using a Western blot with his6 antibodies.