Other
U6-g2

Part:BBa_K2538001:Design

Designed by: Mor Pasi   Group: iGEM18_BGU_Israel   (2018-09-03)


gRNA#2 for SaCas9 targeting of IKKβ under U6 promoter


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]


Design Notes

Designed using Geneious to meet registry standards and with http://chopchop.cbu.uib.no/ to target IKKβ gene. gRNA spacer was cloned into pX601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA was a gift from Feng Zhang (Addgene plasmid # 61591) via Gibson assembly.

Part was cloned into pSB1C3 using EcoRI and PstI restriction sites.



Source

Sequence of gRNA: Designed using http://chopchop.cbu.uib.no/ to target IKKβ gene in the mouse genome. (Mouse GRCm38/mm10)

Sequence of U6 promoter and gRNA SaScaffold: Ran FA, Cong L, Yan WX, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520(7546):186-191. doi:10.1038/nature14299..‏ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4393360/?report=classic


References

1. Frakes, A. E., Ferraiuolo, L., Haidet-Phillips, A. M., Schmelzer, L., Braun, L., Miranda, C. J., ... & Popovich, P. G. (2014). Microglia induce motor neuron death via the classical NF-κB pathway in amyotrophic lateral sclerosis. Neuron, 81(5), 1009-1023.‏

2. Ran, F. A., Cong, L., Yan, W. X., Scott, D. A., Gootenberg, J. S., Kriz, A. J., ... & Koonin, E. V. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature, 520(7546), 186.‏

3. Liddelow, S. A., Guttenplan, K. A., Clarke, L. E., Bennett, F. C., Bohlen, C. J., Schirmer, L., ... & Wilton, D. K. (2017). Neurotoxic reactive astrocytes are induced by activated microglia. Nature, 541(7638), 481.‏