Part:BBa_K5062030
Cas9-GFP
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 3643
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 2740
Illegal NgoMIV site found at 3649 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 4754
Illegal SapI site found at 3768
Illegal SapI.rc site found at 1159
Illegal SapI.rc site found at 1401
Overview
Cas9 (Csn1) endonuclease originates from the Streptococcus pyogenes Type II CRISPR/Cas system (Xu et al., 2019), assembled with GFP from Aequorea victoria. Cas9 can be used with a guide RNA to cleave genomic DNA at a specified location and is a common tool in gene editing, while GFP is used as a fluorescent marker and can be used to track the localization and presence of labelled molecules (Chudakov & Lukyanov, 2003). Both Cas9 and GFP are placed within the same reading frame to assemble a Cas9-GFP fusion protein, which has been shown to increase the mutagenesis and efficiency of the Cas9 endonuclease, without a significant increase in off-target effects (Park et al., 2021).
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.
The action of Cas9 (Ding et al., 2016)
References
Chudakov, D. M., & Lukyanov, K. A. (2003). Use of green fluorescent protein (GFP) and its homologs for in vivo protein motility studies. Biochemistry (Moscow), 68(9), 952–957. https://doi.org/10.1023/a:1026048109654
Ding, Y., Li, H., Chen, L., & Xie, K. (2016). Recent advances in genome editing using CRISPR/CAS9. Frontiers in Plant Science, 7. https://doi.org/10.3389/fpls.2016.00703
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\
Park, J., Yoon, J., Kwon, D., Han, M.-J., Choi, S., Park, S., Lee, J., Lee, K., Lee, J., Lee, S., Kang, K.-S., & Choe, S. (2021). Enhanced genome editing efficiency of CRISPR PLUS: Cas9 chimeric fusion proteins. Scientific Reports, 11(1), 16199. https://doi.org/10.1038/s41598-021-95406-8
Xu, X., Wan, T., Xin, H., Li, D., Pan, H., Wu, J., & Ping, Y. (2019). Delivery of CRISPR/Cas9 for therapeutic genome editing. The Journal of Gene Medicine, 21(7). https://doi.org/10.1002/jgm.3107
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