Part:BBa_K3752000

OXA-48 Parent cgRNA

cgRNA which senses the mRNA of OXA-48 - used as a riboswitch by 2021 Warwick iGEM.

Sequence and Features

NUPACK modelling was used to confirm the function of this part in silico as laboratory work could not be used to verify this due to time constraints.

As seen above, the natively folded state of the cgRNA does not present the dCas9 binding handle.

Yet when the cgRNA anneals with the OXA-48 mRNA, the dCas9 binding handle (top in this image) is folded correctly, switching the CRISPRa system on.

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]

Below is a NUPACK simulation of the folding of this cgRNA (centre) as opposed to a cgRNA with a shorter sensing loop (left) and one with a longer sensing loop (right). All sensing loops labelled in yellow.

The cgRNA with the shorter sensing loop fails to fold into the correct secondary structure at all, making it unable to function at all. The cgRNA with the longer loop folds correctly but the longer sensing domain reduces specificity, making cross-activation more likely.

Above is a simulation of the fluorescence obtained through the use of these cgRNAs; as expected, the shorter sensing loop causes no increase in fluorescence with respect to the negative control, whereas both the optimum length and long cgRNAs offer a marked increase in the expression of the fluorescent reporter.

Above is a comparison of the simulated folding of the optimum cgRNA and a cgRNA with a central mismatch when compared to BBa_K3752000. The probability of the mismatching cgRNA equilibrating in the right conformation is low (20%), making its activating effect far lower than the optimum one's. The comparison in fluorescence between the two is visible below.

Below is a comparison between the cgRNA with the shorter sensing loop described previously and a cgRNA with a mismatch at one of the ends of the sensing loop. Both tend to adopt similar secondary structures, which are known to be inactive - the cgRNA can therefore tolerate no mutations towards the ends of its sensing loop.

This is verified through a simulation, the graph for which is visible below.