Part:BBa_K2330000:Design

How this part was made:

The sequence of the SaCas9 gene was originally taken from the biobrick BBa_K2019000produced by the iGEM16_Northwestern. We planned to deliver our SaCas9 using the P1 phage. To do so, the SaCas9 sequence was re-design to allow its use with P1 phage. We have adopted a fast, reliable and easy method to insert/eliminate the spacers (not included in the BioBrick) and the SaCas9 was further re-designed in accordance to the system [2]. Four illegal restriction enzymes site (ApaI, BsaI, BsiWI, AvrII) were removed without changing the amino acids code. The strategy to insert the spacers relied on the type IIS restriction enzyme BsaI. A BsaI-specific region was designed downstream SaCas9 and between two repeats: the enzymes had its binding site inside this region but cut the right and left extremities of the repeats producing two different but unique overhangs. In this way, it was possible to replace the BsaI-specific region with any desired spacer construct which had BsaI sites and the compatible overhangs. In addition, to make synthesis easier, the SaCas9 was divided into two parts, SaCas9 Part I and Part II. The parts could be connected by ApaI without creating any scar, frameshift or changing the amino acid. The ApaI site was generated by changing the third codon of G881. The finalised sequence was synthesised by IDT.

As already mentioned, four restriction sites were essential for our design. We have digested/ligated the SaCas9 Part I into the P1 phagemid (the essential plasmid containing the P1 phage genes allowing us to produce viable P1 particles. From Dr. Russell Brown, unpublished data). To do this, we used BsiWI and AvrII and we have then digested/ligated Part II with Part I using ApaI and AvrII (Figure 1).

Figure 1: The Cas constructs were divided into Part I and Part II. Once each Part I (top) was inserted in the backbone (not shown) with BsiWI and AvrII, each Part II (bottom) was introduced into Part I using HindIII/ApaI (for Cpf1 and SaCas9 respectively) and AvrII as shown.

How to use the part:

To use this part, you need to design/choose five genetic parts, listed below and in Figure 2:

-Promoter: Staphylococcus aureus native promoter is preferred -tracrRNA: sequence available in [1]. Essential to link SaCas9 with the guiding RNA -RBS -Direct repeat: sequence available in [1] -Spacer: 21bp spacer must be chosen from the target DNA sequence, which is followed by PAM sequence 5’-NNGRRT-3’ [1] -Terminator

Be careful: the SaCas9 sequence contains a His-tag at the 5'!!

Figure 2: Example of SaCas9 cassette

References

     [1] Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., Zetsche, B.,  
     Shalem, O., Wu, X., Makarova, K. S., Koonin, E. V.  Sharp, P.A., Zhang, F. 2015. In vivo
     genome editing using Staphylococcus aureus Cas9. Nature. 520 (7546). pp.186-191.

     [2] Addgene Blog: https://media.addgene.org/cms/files/pCas9_protocol.pdf, written by Marcy Patrick