Part:BBa_K2538002

gRNA#5 for SaCas9 targeting of IKKβ under U6 promoter

gRNA for SaCas9 targeting the 4 exon (genomic location: chr8:22693841) of mouse IKKβ (Inhibitor of nuclear factor kappa-B kinase subunit beta ) gene under the control of human U6 (RNA polymerase III) promoter.

This gRNA is a candidate for IKKβ gene knockout which will inhibit cytokine secretion in the NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) pathway.[1]

Usage and Biology

Guide RNA (gRNA) is a hundred base-long molecule, it includes a scaffold sequence which has a unique two dimensional structure necessary for Cas9 binding and a spacer sequence designed to target a specific location in the genome. gRNA scaffold sequence for SaCas9 was used.[2]

In the NF-κB pathway, IKKβ phosphorylates the IkB inhibitory protein, a process which enables the release of NF-κB to the nucleus where it binds to its cognate DNA sequences to induce gene expression of several cytokines such as IL-1α and TNF.[1] These cytokines have a crucial role in the induction of A1 reactive astrocytes. By knocking out the IKKβ gene we could reduce the IL-1α and TNF secretion from microglia cells and reduce the amount of reactive astrocytes by preventing the formation of their reactive form.[3] By reducing the amount of reactive astrocytes we will prevent motor neuron death and prolong ALS patient survival.

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.‏

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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]