RNA

Part:BBa_K1559000:Design

Designed by: Xiuqi (Rex) Xia   Group: iGEM14_Toronto   (2014-10-07)

tracr RNA to be used with Cas9


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Source

pCas9

We obtained our pCas9 sample by way of Part:BBa_K1218011 (not annotated)

Original pCas9 plasmid, available from addgene: http://www.addgene.org/42876/

pCas9 sequence in genbank format (Our annotations are sourced from here):

http://www.addgene.org/static/data/42/17/509f1fc8-85d2-11e2-92ba-003048dd6500.gb

Email reply from Wenyan Jiang:

The type II CRISPR-Cas system, i.e. tracr, Cas9 and crRNA array was directly cloned from s.pyogenes, with a modification of the array to facilitate the cloning of spacers as you know. I’m not sure which unannotated region of pCas9 you were referring to, perhaps from nucleotides 2015 to 2224? This region contains a bi-directional promoter that drives the expression of both tracr and cas9. And of course they were cloned directly from s. pyogenes without any engineering.

Email reply from Luciano Marraffini:

We did not engineer any promoter or terminator. The sequences are those present in the crispr system of Streptococcus pyogenes and work constitutively in E. coli

Terminators

Terminator/stem-loop prediction was done using ARNold:

http://rna.igmors.u-psud.fr/toolbox/arnold/index.php

References

Jiang, W., Bikard, D., Cox, D., Zhang, F., & Marraffini, L. A. (2013). RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature Biotechnology, 31(3), 233 – 239. doi:10.1038/nbt.2508