Part:BBa_K1723011

c3_2 gRNA expressing sequence

The c3_2 guide RNA (gRNA) consists of the 20 base pair specificity determinant sequence (SDS) c3_2 (ATATACCCCTGCATACCCTA) followed by a gRNA scaffold that stabilizes the RNA complex. The gRNA is flanked by two self-cleaving ribozymes: a Hammerhead Ribozyme and a Hepatitis Delta Virus (HDV) Ribozyme thus freeing the gRNA from the rest of the RNA transcript, and making it possible to produce the gRNA using RNA Polymerase II [1]. Then, the gRNA can form a complex with dCas9-VP64 (BBa_K1723021) (or theoretically other Cas9 mutants). The complex c3_2-dCas9_VP64 will bind specifically to ATATACCCCTGCATACCCTA sequences in the genome of the host organism situated directly before a PAM sequence (NGG). Binding of the complex on the c3_2 sequence of promoter CYC_2 (BBa_K1723024) will result in activating CYC_2 via recruitment of the RNA polymerase II by the VP64 subunit of dCas9_VP64. A similar mechanism has been proven to work with gRNA c3_0 (produced by BBa_K1723009) and promoter CYC_0 (BBa_K1723022) [2].

To learn more about how this part was used specific to our project, please follow this link: http://2015.igem.org/Team:EPF_Lausanne/Part_Collection

Usage and Biology

S. cerevisiae