Part:BBa_K5490021:Design
gRNA ARRAY FOR CASRX , SPACER 1,2,3 (WNV)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We designed a composite array consisting of three spacer regions targeting West Nile Virus (WNV) and two direct repeat (DR) sequences; the other two DRs at the front and back will be provided by the vector. All DR sequences are 36 base pairs in length, except for the last one in the insert, which is 30 base pairs. This adjustment was made as a compromise to reduce the complexity of the insert, facilitating its ordering from the supplier.
Additionally, we introduced BbsI enzyme sites at the borders of the insert, with the cleavage sites facing each other. The chosen vector, pSLQ5429_pUC_hU6-crScaffold_EF1a-BFP, serves as the expression backbone for the gRNA array and contains additional DR sequences downstream of the multi-linker, also incorporating BbsI sites. This design introduces an additional maturation step, enhancing the stability of the gRNA array. Moreover, it prevents the last gRNA from forming complexes with RNAs in the absence of CasRx, thereby providing us with more precise control over the system.
Source
Source:Silvana Konermann Laboratory of Molecular and Cell Biology, Salk Institute for Biological Studies, 10010 N Torrey Pines Rd, La Jolla, CA 92037, USA Direct repeat sequence,firstly identified at Ruminococcus Spacer sequence desingned by iGEM IOANNINA 2024 dry lab team