Designed by: Isabella Loft Group: iGEM16_DTU-Denmark (2016-10-14)
SCR1'-tRNA
This basic part encodes the gRNA promoter SCR1_tRNA.
synthetic RNA polymerase III promoter
441 base pairs
for Yarrowia lipolytica
The SCR1’-tRNA promoter is a synthetic RNA polymerase III promoter. It has been used for CRISPR-Cas9-mediated gene disruption in Y. lipolytica with good results.
The promoter was amplified from pCRISPRyl using (PCR) with primer tails encoding prefix10 and suffix10. In these primers we also introduced a base substitution to eliminate an illegal restriction site and by that making the part compatible with the iGEM biobrick standards.
We have proved the functionality of this part in Y. lipolytica, as we observed Cas9 activity of a CRISPR-Cas9 plasmid (pIW357) that contained this SCR1’-tRNA promoter. We tested pIW357 in the Y. lipolytica PO1fΔKu70 strain. In order to account for Cas9 activity of pIW357, we also transformed pCRISPRyl as a positive control (pCRISPRyl does not contain a functional protospacer). With an intact NHEJ repair pathway and/or a template for HR, the induced DSB should easily be repaired. However, as this is the PO1fΔKu70 strain and as there is no HR donor template available, the induced DSB should be lethal. Based on the difference in transformant counts between the pIW357 and pCRISPRyl transformations, pIW357 is functioning properly and thus the SCR1’-tRNA promoter is functioning properly, too.
Sequence and Features
