Team:Edinburgh OG/Parts

PhagED: a molecular toolkit to re-sensitise ESKAPE pathogens

Parts

This year the iGEM Edinburgh_OG team focused on developing a modular toolkit using CRISPR systems and phages to re-sensitise antibiotic-resistant bacteria. As a BioBrick we submit the E. coli codon-optimised Staphylococcus aureus Cas9.

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http://www.nature.com/news/genome-editing-revolution-my-whirlwind-year-with-crispr-1.19063

How does this part work?

  • Our SaCas9 can be programmed to cleave specific target sequence followed by the PAM sequence (5’-NNGRRT-3’).
  • To express SaCas9, it requires suitable machinery such as promoter, RBS,and terminator.
  • To programme SaCas9, you need to design guide RNA (tracrRNA [2], 21 bp spacer flanked by direct repeats [2] ).

Advantages of SaCas9 compared with the conventional Streptococcus pyogenes Cas9:

  • Smaller size (1053 amino acids against 1368) resulting in an easier expression/delivery
  • Different PAM sequence recognised (5’-NNGRRT-3’ ) increasing the usability
  • Higher efficiency of SaCas9 over SpCas9 [2]

[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] Friedland AE, Baral R, Singhal P, et al. Characterization of Staphylococcus aureus Cas9: a smaller Cas9 for all-in-one adeno-associated virus delivery and paired nickase applications. Genome Biology. 2015;16:257. doi:10.1186/s13059-015-0817-8.