Part:BBa_K5102018

gRNA (30bp spacer) for switching state 0 to 1 for ProgRAM (tape 1)

The part encodes a guide RNA (gRNA) that functions to switch the state from 0 to 1 in the ProgRAM recorder. This gRNA directs a deactivated Cas13b protein from the Prevotella species, which is fused to the ADAR2 deaminase domain. The Cas13b-gRNA complex guides the deaminase to the target sequence, facilitating precise RNA editing for state change.

During recording tape design part design careful considerations have been taken to ensure high editing rates, as well as specificity of gRNA binding. The molecular recording utilized in the project is based on the REPAIR v2 system developed by Cox et al., 2017. It has previously been shown that both the nucleotides surrounding the modified adenosine, called central base triplet (CBT), as well as location of the modified A within the sequence play an important role in the editing efficiency. For 30 nucleotide gRNA spacer design, 22nd and 28th positions have been shown to be the most efficient. Introduction of a A12G mismatch showed increased binding disruption of gRNA, ensuring highly selectable editing. While several Cas enzymes require a highly conserved Protospacer adjacent motif (PAM) sequence, PspCas13b does not require it for its activity. However, it still shows a preference for targets with protospacer flanking sites (PFSs).

After applying all of these considerations, we were left with the following minimal tapes design:

The missing nucleotides were designed in a way that: (i) N could be replaced by any nucleotide but adenosine, (ii) the gRNA complementary to the binding tape must have formed a conserved stem loop structure required by PspCas13b, which binds the ADAR2DD to the target deamination site. To ensure best design, to this end we have employed computational methods.

References

Cox, D. B. T., Gootenberg, J. S., Abudayyeh, O. O., Franklin, B., Kellner, M. J., Joung, J., & Zhang, F. (2017). RNA editing with CRISPR-Cas13. Science, 358(6366), 1019–1027. https://doi.org/10.1126/science.aaq0180

Sequence and Features

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]