Coding

Part:BBa_K1699004

Designed by: Emil Ruvinov   Group: iGEM15_BGU_Israel   (2015-08-09)

gRNA for SaCas9 targeting human ubiquitin B gene under U6 promoter

gRNA for SaCas9 targeting human ubiquitin B gene (UBB) under the control of human U6 (RNA polymerase III) promoter. gRNA sequence is complementary to 3 different loci in the second exon of human UBB gene, which is essential for cancer cells (1, 2).


Usage and Biology

Guide RNA is a hundred base-long molecule with a unique two dimensional structure which binds Cas9 and guides it to a dsDNA sequence complementary to 21-22 base pairs on the 5' end of the molecule. gRNA scaffold sequence for SaCas9 was used (3). UBB gene was chosen as a model for SaCas9-mediated gene knock-out. UBB is essential for survival of multiple cancer cell types (1, 2).

Characterization

This part was designed as a control construct for two cancer-specific promoter-based design of CRISPR-mediated gene knock-out. In the design, gRNA is ribozyme-flanked (to enable synthesis under RNA polymerase II promoter) and is under control of human survivin promoter.

U6-gUBB: AAV vector for the synthesis of gRNA for SaCas9 targeting human Ubiquitin B gene (abbreviated as gUBB) under the control of human U6 promoter (Fig. 1).

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Fig. 1. Plasmid map of AAV vector carrying gUBB under the control of human U6 promoter.

References

1. Downregulation of ubiquitin level via knockdown of polyubiquitin gene Ubb as potential cancer therapeutic intervention. Oh C, Park S, Lee EK, Yoo YJ. Sci Rep. 2013;3:2623. doi: 10.1038/srep02623. http://www.ncbi.nlm.nih.gov/pubmed/24022007

2. The ubiquitin-proteasome system as a prospective molecular target for cancer treatment and prevention. Chen D, Dou QP. Curr Protein Pept Sci. 2010 Sep;11(6):459-70. Review. http://www.ncbi.nlm.nih.gov/pubmed/20491623

3. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. http://www.ncbi.nlm.nih.gov/pubmed/25830891

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 250
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
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