Part:BBa_K5062045
Multiplex Cas12a guide RNA (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 anti-SIRPa guide RNA (gRNA) (BBa_K5062022), anti-PD1 gRNA (BBa_K5062024), anti-VTCN1 gRNA (BBa_K5062026) and STAB1 gRNA (BBa_K5062028) to allow for multiplexed Cas12a-mediated CRISPR operations. 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 shRNA 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 mulitple base-perfect shRNA.
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 Cas12a mechanism works similarly to Cas9, with the difference of four separate guide RNAs composed of only the crRNA, thus being able to knock out up to 4 targets, rather than just 1 target with Cas9 (Anvar et al., 2024). In our design, aside from the SIRPα domain which is a crucial immune evasion checkpoint (Ray et al., 2018), three other targets have been chosen to increase the efficacy of our CAR macrophage. The PD1-PDL1 pathway, which acts as a T-cell activation checkpoint, and immune evasion checkpoint (Feng et al., 2019). STAB1, which involves inhibition of phagocytosis (Hollmén et al., 2020). As well as the VTCN1 which plays a role in T-cell inhibition (Podojil & Miller, 2017). Targeting these domains allows our macrophage to increase phagocytic ability and boost T-cell activity, overall targeting immune evasion with an increased efficacy.
A diagram depics the differnces between Cas9 and Cas12a (Froehlich, J. J., 2024)
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
Anvar, N. E., Lin, C., Ma, X., Wilson, L. L., Steger, R., Sangree, A. K., Colic, M., Wang, S. H., Doench, J. G., & Hart, T. (2024). Efficient gene knockout and genetic interaction screening using the in4mer CRISPR/Cas12a multiplex knockout platform. Nature Communications, 15(1). https://doi.org/10.1038/s41467-024-47795-3
Feng, M., Jiang, W., Kim, B. Y. S., Zhang, C. C., Fu, Y., & Weissman, I. L. (2019). Phagocytosis checkpoints as new targets for cancer immunotherapy. Nature Reviews. Cancer, 19(10), 568–586. https://doi.org/10.1038/s41568-019-0183-z
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
Froehlich, J. J. (2024, August 18). Cas12a and Cas9 nucleases and their DNA cleavage positions. https://en.wikipedia.org/wiki/Cas12a#/media/File:Cas12a_vs_Cas9_cleavage_position.svg
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
Hollmén, M., Figueiredo, C. R., & Jalkanen, S. (2020). New tools to prevent cancer growth and spread: a ‘Clever’ approach. British Journal of Cancer, 123(4), 501–509. https://doi.org/10.1038/s41416-020-0953-0
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
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
Podojil, J. R., & Miller, S. D. (2017). Potential targeting of B7‐H4 for the treatment of cancer. Immunological Reviews, 276(1), 40–51. https://doi.org/10.1111/imr.12530
Ray, M., Lee, Y., Hardie, J., Mout, R., Tonga, G. Y., Farkas, M. E., & Rotello, V. M. (2018). CRISPRed macrophages for Cell-Based Cancer immunotherapy. Bioconjugate Chemistry, 29(2), 445–450. https://doi.org/10.1021/acs.bioconjchem.7b00768
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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