Part:BBa_K5062039
Anti-SIRPa Cas9 gRNA Release System
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Overview
This is a release system designed for the release of Cas9 guide RNA (gRNA) (BBa K5062020) to allow for CRISPR to disrupt the expression of SIRPa. The system utilizes a 5' Hammerhead Ribozyme (BBa_K5062009) and a 3' Hepatitis Delta Virus (HDV)(BBa_K5062010) Ribozyme to catalytically self-cleave any unnecessary sequences at 5' and 3' ends of the shRNA allowing for its exact sequence to be preserved.
This system allows gRNA expression using any RNA polymerase, including those that inherently contain capping and tailing capabilities such as the RNA polymerase of Venezuelan equine encephalitis (VEE) virus which powers self-replicating RNA (srRNA) systems, allowing a transient srRNA system to sustain the subgenomic expression and release of base-perfect gRNA.
In vitro characterisation of this part is currently ongoing prior to the wiki freeze. Full part characterisation is expected by the 2024 iGEM Giant Jambouree.
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.)
The self-cleavage reaction catalyzed by the Hammerhead Ribozyme involves a phosphodiester isomerization mechanism. This results in the formation of a 2',3'-cyclic phosphate at the 5' end and a 5'-hydroxyl group at the 3' end of the cleaved RNA. The reaction is reversible, allowing the Hammerhead Ribozyme to also catalyze the ligation of the cleaved RNA strands (Hammann et al., 2012).
The Hammerhead Ribozyme is considered one of the best-characterized ribozymes. Its small size, well-studied cleavage chemistry, known crystal structure, and biological relevance make it a useful model system for understanding the fundamental mechanisms of RNA catalysis. Hammerhead Ribozymes have been found ubiquitously across all domains of life, where they play roles in processes like rolling-circle replication of viral genomes and co-transcriptional processing of retrotransposons (Jimenez et al., 2015).
The sequence is only the core of the hammerhead ribozyme. A 6 nucleotide head should be added to the 5' end of the ribozyme core which is complementary to the first 6 nucleotides of the RNA to be released to allow a succesful cleavage. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.
A diagram depics the design considerations required for using the 5' Hammerhead Ribozyme (UC San Diego, n.d.)
The HDV ribozyme has a characteristic nested double-pseudoknot secondary structure with five helical regions (P1, P1.1, P2, P3, P4) (Ferré-D’Amaré & Scott, 2010). The HDV ribozyme catalyzes a site-specific transesterification reaction, where the 2' hydroxyl group of the nucleotide upstream of the cleavage site acts as a nucleophile to attack the adjacent phosphodiester bond. This results in the formation of a 2'-3' cyclic phosphate and a 5' hydroxyl product (Jimenez et al., 2015).
The HDV ribozyme can function through both metal-dependent and metal-independent mechanisms. In the metal-dependent mechanism, a divalent metal ion (typically Mg2+) helps to stabilize the developing negative charge on the 2' nucleophile and the pentavalent phosphorane transition state (Ke et al., 2004). In the metal-independent mechanism, the catalytic cytosine (C75) can act as a general acid to protonate the 5' leaving group (Ferré-D’Amaré & Scott, 2010).
The self-cleavage activity of the HDV ribozyme is essential for processing the HDV genomic and antigenomic RNA strands during rolling-circle replication (Ferré-D'Amaré, A.R et. al,2010). The cleavage generates linear RNA strands with 5' hydroxyl and 2'-3' cyclic phosphate ends, which can then be ligated to form the circular HDV genome (Jimenez, R.M. et al., 2015).
This can be added directly to the 3' end of RNA sequences. Since this is a DNA part, it should be added to the DNA that eventually transcirbes into the relevant RNA sequences.
A diagram depics the design considerations required for using the 3' HDV Ribozyme (UC San Diego, n.d.)
References
Ferré-D’Amaré, A. R., & Scott, W. G. (2010). Small self-cleaving ribozymes. Cold Spring Harbor Perspectives in Biology, 2(10), a003574. https://doi.org/10.1101/cshperspect.a003574
Hammann, C., Luptak, A., Perreault, J., & De La Peña, M. (2012). The ubiquitous hammerhead ribozyme. RNA, 18(5), 871–885. https://doi.org/10.1261/rna.031401.111
Jimenez, R. M., Polanco, J. A., & Lupták, A. (2015). Chemistry and Biology of Self-Cleaving Ribozymes. Trends in Biochemical Sciences, 40(11), 648–661. https://doi.org/10.1016/j.tibs.2015.09.001
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
Ke, A., Zhou, K., Ding, F., Cate, J. H. D., & Doudna, J. A. (2004). A conformational switch controls hepatitis delta virus ribozyme catalysis. Nature, 429(6988), 201–205. https://doi.org/10.1038/nature02522
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
UC San Diego. (n.d.). 3’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/3_prime_ribozyme_design.html
UC San Diego. (n.d.). 5’ Ribozyme design. https://labs.biology.ucsd.edu/zhao/CRISPR_web/5_prime_ribozyme_design.html
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