RNA
SPACER3WNV

Part:BBa_K5490003:Design

Designed by: IOANNIS VASILEIOS ELAFROPOULOS   Group: iGEM24_IOANNINA   (2024-09-23)
Revision as of 00:52, 24 September 2024 by Tzonissss13 (Talk | contribs) (→‎Design Notes)


23-nt sequence binds CasRx to cleave WNV genome; modifiable target 3


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Design Notes

The gRNAs are complex in their design, particularly because they are part of a composite system. Each gRNA is expressed as a polycistronic RNA, which, upon expression, interacts with the CasRx protein. This interaction leads to the maturation of the gRNA and its subsequent cleavage into three distinct gRNAs, each targeting different regions of the virus. Given this complexity, it is crucial to validate the functionality of each individual gRNA before assembling them into the composite array. The gRNA array consists of two main components: Direct Repeats (DRs): These sequences produce secondary structures that are essential for the CasRx protein to bind, initiating the maturation process of the gRNAs. Spacer Sequences: These sequences are complementary to specific regions of the virus and guide the endonuclease activity of CasRx to its target. By validating each gRNA separately, we ensure that each component of the array functions correctly before combining them into the final composite part. This step is essential for achieving precise targeting and effective cleavage of the viral genome. First, for the individual gRNAs, we ordered three separate inserts, each consisting of a direct repeat (DR) sequence upstream a specific spacer sequence downstream. These inserts were designed with recognition sites for the Type IIS restriction enzyme BbsI, positioned at both the 5' and 3' ends. This design ensures that after cleavage, the recognition sites are removed, leaving only the desired gRNA sequence. The BbsI recognition sites were placed facing each other to facilitate this separation.

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


References