From 2013.igem.org

Background and Enabling Technologies

The 2013 iGEM team engineered an exosome mediated cell-cell communication system. Exosomes are 30-100nm extracellular vesicles that contain miRNAs, mature mRNAs and proteins. Discharged from sender cells via exocytosis, exosomes fuse to the target cell membrane and release their contents into the receiver cell. In 2011 Steve Gould’s lab showed that fusing GFP to the TyA protein and adding an N-terminal acylation tag led to protein oligomerization and selective targeting to the protein membrane, facilitating exosome mediated export of GFP. (Shen, 2011). We hope to engineer sender cells that export proteins of interest, by fusing them to Acyl-Tya.

Our project also utilizes the Cas9/CRISPR system. Cas9, a nuclease derived from bacteria can be directed to target specific DNA sequences by guide RNAs that are complementary to target DNA sequences. (Barrangou, 2007). We employ a mutated version of Cas9 that simply binds the target sequence rather than making an incision. Because the Cas9/ CRISPR system can be easily manipulated to target virtually any region in the genome, Cas9 has many potential medical applications. For example, Cas9 can be used to target and modify specific DNA sequences through nuclease activity (Barrangou, 2007). As a DNA sensor, Cas9 can be used to screen cells for specific mutations. Finally, Cas9 could also be fused to transcription factors and chromatin modifiers, allowing arbitrary modulation of gene expression. Exosomes could be a means of introducing regulatory proteins to a naïve cell, opening up therapeutic avenues as well.

Citations

Bacchus, William et al. Synthetic two-way communication between mammalian cells. Nat. Biotechnol. 30, 991–996 (2012) Lancaster, Madeline et al. Cerebral organoids model human brain development and microcephaly. Nature 501, 373–379 (2013) Barrangou, Rodolphe et al. CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes. Science 314, 1709-1712 (2007) Shen, B et al. Protein targeting to exosomes/microvesicles by plasma membrane anchors. J Biol Chem. (2011)