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.
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Spacer Sequences: These sequences are complementary to specific regions of the virus and guide the endonuclease activity of CasRx to its target.
Spacer Sequences: These sequences are complementary to specific regions of the virus and guide the endonuclease activity of CasRx to its target.
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
