RNA

Part:BBa_K5062020

Designed by: Alika Yuk Wai Wong   Group: iGEM24_HKU-HongKong   (2024-09-27)


Cas9 gRNA targeting SIRPa

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]


Overview

This part is encodes for a Cas9 gRNA targeting SIRPa. This allows for the Cas9-mediated knockout of SIRPa preventing the expression the SIRPa receptor on the cell surface. This disrupts the SIRPa/CD47 immune evasion pathway increasing phagocytosis of tumor cells.


Usage and Biology

The Cas9 mechanism involves the formation of a Cas9 complex, consisting of the Cas9 protein and a guide RNA (gRNA). The gRNA is composed of the target-specific crRNA, whose sequence is complementary to the target domain, and the scaffolding tracrRNA, which allows for binding with the Cas9 endonuclease. When formed, the gRNA is used to direct the Cas9 enzyme to a specific gene of interest, where the Cas9 enzyme is then able to induce a double-strand break at the specific site, being the SIRPα domain (Kim et al., 2014). With the use of cell’s natural DNA repair pathways, such as non-homologous end joining, the excised DNA is repaired, however, small insertions and deletions (indels) are introduced at the junction. As such, the error-prone DNA sequence results in disrupted protein expression (Ishibashi et al., 2020). The final product is a SIRPα-deficient macrophage, able to interrupt the SIRPα-CD47 pathway and thus preventing the immune evasion of tumor cells, while allowing for phagocytosis and an overall increase in the efficacy of CAR macrophages.


A diagram depics the Cas9-gRNA interaction (The Complete Guide to Understanding CRISPR SGRNA, n.d.)



References

Ishibashi, A., Saga, K., Hisatomi, Y., Li, Y., Kaneda, Y., & Nimura, K. (2020). A simple method using CRISPR-Cas9 to knock-out genes in murine cancerous cell lines. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-79303-0

Kim, S., Kim, D., Cho, S. W., Kim, J., & Kim, J. (2014). Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Research, 24(6), 1012–1019. https://doi.org/10.1101/gr.171322.113\

The Complete Guide to Understanding CRISPR SGRNA. (n.d.). Synthego. https://www.synthego.com/guide/how-to-use-crispr/sgrna



[edit]
Categories
Parameters
None