PARTS

Our part collection consists of two composite parts which are themselves constructed by different combinations of 5 new basic parts. The collection consists of a T7 promoter (BBa_K3457003), RBS (BBa_K4634001), His6-tag (BBa_255Q3II5), OKT4 or gp120 (BBa_25EULV3S), Diphtheria toxin A (BBa_25ML4821), followed by a LshC2c2 protein (BBa_25CD9PFJ). When expressed in E. coli and further processed in a cell-free glycosylation system, the fusion proteins can be evaluated for correct folding and functionality. Two variants were constructed: one containing the OKT4 sequence and another containing the gp120 sequence. These variants enable comparative analysis of cellular uptake and RNA cleavage efficiency, providing insight into the system’s targeting and catalytic performance. Experimental validation is currently in progress, and updated results will be presented at the Paris Jamboree.

NEW BASIC PARTS IN THE COLLECTION

Part Name Part ID Type Functional Description
LshC2c2 BBa_25CD9PFJ Coding When LshC2c2 (Cas13a protein) binds to its guide RNA (gRNA), it recognizes and attaches to complementary ssRNA targets. Upon binding, it cleaves the target RNA and also induces collateral cleavage of nearby ssRNA molecules.
Diphtheria toxin A-chain (DTA) BBa_25ML4821 Protein Domain The diphtheria toxin A-chain functions as the active toxin component within the delivery system, facilitating the endocytosis of the attached domain across the cell membrane. Acidification of the endocytic vesicle induces a conformational change, enabling cytosolic delivery.
Glycoprotein 120 (gp120) BBa_25EULV3S Coding The gp120 molecule is a surface glycoprotein on HIV-1 that binds CD4 receptors on host cells, initiating endocytosis.
Hexa Histidine tag (His tag) BBa_255Q3II5 Coding 6x His tag aids in protein purification and detection via nickel affinity chromatography and western blotting.
Table 1: Table of individual protein domains and coding sequences. Pink shading indicates key functional domains involved in delivery and activity.

NEW COMPOSITE PARTS IN THE COLLECTION

Part Name Part ID Type Functional Description
LshC2c2-DTA-gp120 fusion protein BBa_25TK6V7U Coding The fusion protein will bind to CD4 receptors via the gp120 domain. DTA induces endocytosis of LshC2c2 and DTA into HEK293 cells. Once inside, LshC2c2 may synthesize gRNA and cleave ssRNA within the cell.
LshC2c2-DTA-OKT4 fusion protein BBa_25XDWXJX Coding The fusion protein binds CD4 receptors via the OKT4 domain. DTA enables endocytosis of LshC2c2 and DTA into HEK293 cells. Once inside, LshC2c2 may synthesize gRNA, resulting in cleavage of existent ssRNA.
Table 2: Table of fusion proteins combining multiple domains.

REFERENCES

  1. Abudayyeh, Omar O., et al. “C2c2 Is a Single-Component Programmable RNA-Guided RNA-Targeting CRISPR Effector.” Science, vol. 353, no. 6299, 2016, pp. aaf5573. American Association for the Advancement of Science, https://doi.org/10.1126/science.aaf5573.
  2. Burris, Brandon Joseph Davis, et al. “Optimization of Specific RNA Knockdown in Mammalian Cells with CRISPR-Cas13.” Methods (San Diego, Calif.), vol. 206, 2022, pp. 58–68, https://doi.org/10.1016/j.ymeth.2022.08.007.
  3. East-Seletsky, Alexandra, O’Connell, M. R., Knight, S. C., Burstein, D., Cate, J. H., Tjian, R., and Doudna, J. A. “Two Distinct RNase Activities of CRISPR-C2C2 Enable Guide-RNA Processing and RNA Detection.” Nature, Sept. 2016, https://pubmed.ncbi.nlm.nih.gov/27669025/. DOI: 10.1038/nature19802.
  4. Wang, Z., et al. “CRISPR/Cas9 Can Inhibit HIV-1 Replication but NHEJ Repair Facilitates Virus Escape.” Molecular Therapy, vol. 24, no. 3, 2016, pp. 522–528. Elsevier, https://doi.org/10.1038/mt.2016.24.
  5. Tian, Songhai, et al. “Targeted Intracellular Delivery of Cas13 and Cas9 Nucleases Using Bacterial Toxin-Based Platforms.” Cell Reports, vol. 38, no. 10, Mar. 2022, p. 110476, https://doi.org/10.1016/j.celrep.2022.110476.
  6. “Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein Complex.” RCSB Protein Data Bank, PDB ID: 1GC1, Research Collaboratory for Structural Bioinformatics, 1998. www.rcsb.org/structure/1GC1.
  7. Wan, Yiming, et al. “Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting.” ACS Synthetic Biology, vol. 13, no. 10, 2024, pp. 3212–30, https://doi.org/10.1021/acssynbio.4c00271.
  8. Chirmule, N., et al. “Mechanisms of HIV-1 Entry into CD4+ T Cells.” Current HIV Research, vol. 1, no. 4, 2003, pp. 295–307, https://doi.org/10.2174/1570162033485262.
  9. Greenfield, L., et al. “The Toxic Moiety of Diphtheria Toxin Forms Transmembrane Channels in Planar Lipid Bilayers.” Proceedings of the National Academy of Sciences, vol. 80, no. 3, 1983, pp. 685–689, https://doi.org/10.1073/pnas.80.3.685.
  10. Morris, Garrett M., et al. “AutoDock4 and AutoDockTools4: Automated Docking with Selective Receptor Flexibility.” Journal of Computational Chemistry, vol. 30, no. 16, 2009, pp. 2785–2791, https://doi.org/10.1002/jcc.21256.
  11. Yoon, V., Fridkis-Hareli, M., et al. “Glycoprotein 120.” Current Medicinal Chemistry, https://doi.org/10.2174/092986710790514499.